Compositions comprising nanostructures for cell, tissue and artificial organ growth, and methods for making and using same

ABSTRACT

The invention provides articles of manufacture comprising biocompatible nanostructures comprising nanotubes and nanopores for, e.g., organ, tissue and/or cell growth, e.g., for bone, kidney or liver growth, and uses thereof, e.g., for in vitro testing, in vivo implants, including their use in making and using artificial organs, and related therapeutics. The invention provides lock-in nanostructures comprising a plurality of nanopores or nanotubes, wherein the nanopore or nanotube entrance has a smaller diameter or size than the rest (the interior) of the nanopore or nanotube. The invention also provides dual structured biomaterial comprising micro- or macro-pores and nanopores. The invention provides biomaterials having a surface comprising a plurality of enlarged diameter nanopores and/or nanotubes.

FIELD OF THE INVENTION

The present invention provides articles of manufacture comprisingbiocompatible nanostructures comprising nanotubes and nanopores for,e.g., organ, tissue and/or cell growth, e.g., for bone, tooth, kidney orliver growth, and uses thereof, e.g., for in vitro testing of drugs,chemicals or toxins, or as in vivo implants, including their use inmaking and using artificial tissues and organs, and related diagnostic,screening, research and development and therapeutic uses.

BACKGROUND OF THE INVENTION

It is known that the nano-scaled materials exhibit extraordinaryelectrical, optical, magnetic, chemical and biological properties, whichcannot be achieved by micro-scaled or bulk counterparts. The developmentof nano-scaled materials has been intensively pursued in order toutilize such properties for various technical applications includingbiomedical and nano-bio applications.

Ti and Ti alloys are corrosion resistant, light, yet sufficiently strongfor load-bearing, and are machinable. They are one of the fewbiocompatible metals which osseo-integrate (direct chemical or physicalbonding with adjacent bone surface without forming a fibrous tissueinterface layer). For these reasons, they have been used successfully asorthopaedic (orthopedic) and dental implants. See Handbook ofbiomaterial properties, ed. J. Black and G. Hasting, London: Chapman &Hall, 1998; Ratner et al., Biomaterials Science, San Diego, Calif.,Academic press, 1996.

The bioactivity of Ti, such as the relatively easy formation ofhydroxyapatite type bone mineral on Ti is primarily caused by theoccurrence of Ti oxide on the surface of Ti and its alloys. Among thevarious crystal structures of Ti oxide, the anatase phase is known to bebetter than the rutile and other phases. See, e.g., Uchida (2003) J.Biomedical Materials Res. 64:164-170. Surface treatments such asroughening by sand blasting, formation of anatase phase TiO.sub.2,hydroxyapatite coating, or other chemical treatment have been utilizedto further improve the bioactivity of Ti surface and enhance bonegrowth.

While the fabrication of vertically aligned TiO.sub.2 nanotubes on Tisubstrate was demonstrated by anodization process, an investigation ofsuch titanium oxide nanotubes for bone growth or other bio applicationhas not been attempted. An investigation of such titanium oxidenanotubes for bone growth type bio application has only recently beenreported, showing a significantly enhanced bone growth on TiO.sub.2nanotube array structure. See, e.g., Oh (2005) “Growth of Nano-scaleHydroxyapatite Using Chemically Treated Titanium Oxide Nanotubes”,Biomaterials 26:4938-4943. Patients who go through Ti implant operationsfor repair of hip joints, broken bones, or dental implants often have towait for many months of slow bone growth recovery before they are curedenough to get off the confinement on a bed or crutches and have a normallife. Accelerated bone growth would thus be very beneficial for suchpatients.

The structure of the anodized TiO.sub.2 nanotube array, such as thediameter, spacing and height of nanotubes, is not always easy to controlduring the electrochemical anodization process of pore formation. Forexample, the largest reported diameter of TiO.sub.2 nanotubes is lessthan approximately (about) 100 to 150 nm. While a portion of filopodia,the thin branches of growing cells, can get into such a small pores andenhance cell adhesion-growth, the approximately 100 nm regime ofdimension is too small to accommodate the main part of typicalosteoblast and many other cells as these have a much larger dimension ofmicrometers. In addition, the desired insertion of biological agentssuch as biomolecular growth factors, cytokines, collagens, antibiotics,antibodies, drug molecules, small molecules, inorganic nanoparticles,etc. within the pores for further accelerated cell/bone growth or formedical therapeutics can be facilitated if the inner diameter of thepores are made somewhat larger. Therefore, an ability to artificiallydesign and construct a biocompatible nanostructure, e.g., with aspecific desired nanotube diameter, nanopore dimension and spacing, isdesirable for further controlled and accelerated growth of bones andcells. For orthopaedic and dental applications, a dual structure oflarger dimension pores, which in one aspect can be of re-entrant shape,in combination of nanostructured surface would be desirable to have bothaccelerated cell/bone growth and physically locked-in bone configurationin the re-entrant large pores for improved mechanical durability ontensile or shear strain. Furthermore, if such a biocompatiblenanostructure can be made to easily accommodate biological agent storagein the nano/micro pores to enhance multifunctional roles to additionallyaccelerate bone and cell growth, its practical usefulness can be muchenhanced for various biomedical applications.

Coating of bioactive materials such as hydroxyapatite and calciumphosphate on Ti surface is a commonly used technique to make the Tisurface more bioactive for bone growth purposes. See, e.g.,Shirkhanzadeh (1991) J. Materials Science Letters volume 10; de Groot(1987) J. Biomedical Materials Res. 21:1375-1381; Cotell (1992) J. ofApplied Biomaterials 8:87-92. However, the fatal drawback of thesecurrently available coating techniques is that such a flat andcontinuous coatings tend to fail by fracture or de-lamination at theinterface between the implant and the coating as an adhesion failure, orat the interface between the coating and the bone, or at both boundaryinterfaces. Thick film coatings tends to introduce more interfacestresses at the substrate-coating interface, especially in view of thelack of strong chemical bonding or the absence of common elements sharedby the substrate (e.g., Ti implant) and the coating material. See, e.g.,Yang (1997) J. Biomedical Materials Res. 36:39-48. It would thus bedesirable if the interface is bonded with an improved and integratedstructure, for example, with a locked-in configuration with a muchincreased adhesion area, and as a discrete, less continuous layer tominimize interface stress and de-lamination.

An additional, worthy consideration of bone growth/repair implants isthe ability of the implants to withstand a tensile or shear stress,which tends to break off the interface bonding between the implant andthe bone that is allowed to grow on the implant surface. It would thusbe desirable if the surface geometry of the implant is improved so thatnot only nanoscale interfacial adhesions occur, but microscale andmacroscale lock-in structure is provided to guard against slippage ofthe implant on tensile stress or breakage of the bond on shear stress.

Accelerated cell growth is also desirable not only for bones but alsofor a variety of cells including liver cells, kidney cells, blood vesselcells, skin cells, periodontal cells, stem cells, and so forth. Liver inhuman body is the largest gland and a dynamic organ which serves severalimportant functions, working closely with many fundamental biologicalsystems and bio-processes in the body. The liver is like the mainchemical factory and food storehouse in human body, as it helps the bodydigest food and help purify the blood of the poisons and wastes. Thecomplex functions associated with the liver include; (a) The regulationof blood glucose level, lipids and amino acids, (b) The production andsecretion of bile, red blood cells, blood proteins (such as albumin,globulin, fibrinogen), cholesterol, and glucose, (c) The purification ofblood by removing toxins, wastes, unnecessary hormones, and hemoglobinmolecules, (d) The storage of blood, vitamins and minerals.

The parenchymal cells known as hepatocytes are the major cells populatedin the liver. In additions, several other cells such as endothelialcells, adipocytes, fibroblastic cells and Kupffer cells are alsoincluded in the liver.

A significant portion of the human population (e.g., about one in tenpeople) has been afflicted with liver diseases such as hepatitis, livercancer, and acute or chronic liver failure. Although livertransplantation is an optional treatment method, there is a very limitedsupply of donor organs, and the medical and associated costs for thetransplant procedure and post-operation immunosuppressive drug therapyare considerable.

Many research investigations related to liver cell culture in vitro havebeen conducted to figure out the problem often caused by long-termculture of liver cells. Cultured liver cells can be useful forhepatocytes transplantation, implantable constructs and bioreactorproduction. The primary cultures of rat hepatocytes have beenextensively used to research the effects of potential toxins on enzymeleakage, metabolism, and cellular membranes. See, e.g., Grisham (1979)International Review of Experimental Pathology 20:123-210; Acosta (1981)Biochemical Pharmacology 30:3225-3230. However, there are a number ofknown drawbacks about long-term liver cell culture as some loss of liverfunction is frequently observed. So far, there has been no successfulmeans of proliferating healthy liver parenchymal cells.

In vitro culture of adult hepatocytes does not show prolonged ability toproduce albumin and display cytochrome P-450 enzyme activity. Insuspension culture, the viability of hepatocytes and their cytochromeP-450 enzyme activity declines gradually as a function of incubationtime. In addition, cell division usually is limited to the first 24-48hr of culture after which the cell division is no longer significant.See, e.g., Sirica (1980) Pharmacology Review 31:205-228; Clayton (1983)Molecular and Cellular Biology 3:1552-1561; Chapman (1973) J. CellBiology 59:735-747. In a two-dimensional culture system, the viabilityof adult hepatocytes adhered to the culture plate show somewhat longeractivity periods than other culture systems, but the functionality ofhepatocytes decreased rapidly. See, e.g., Deschenes (1980) In Vitro16:722-730.

To improve hepatocyte growth and prolong liver-specific functions invitro, various kinds of matrices have been studied, such as type I andIV collagen substrates, homogenized liver biomatrix (see, e.g., Reid(1980) Ann. N.Y. Acad. Sci. 349:70-76), sandwich-shaped collagensubstrate composed of two layers of type I collagen, and fibronectincoated plates. See, e.g., Michalopoulos (1975) Experimental Cell Res.94:70-78, Bissell (1987) J. Clinical Investigation 79:801-812; Dunn(1989) FASEB J. 3:174-177; Deschenes (1980) In Vitro 16:722-730. Eventhough many of these experimental approaches have demonstrated anextended viability of hepatocyte and the stability of liver specificfunction under in vivo environment, they are still not satisfactoryenough for practical applications.

An alternative way, which allows liver cells to possess some long-termviability and liver-specific functionality, utilized co-culturing liverparenchymal cells with a diversity of structurally supportive,non-parenchymal stromal cells or non-hepatic stromal cells. See, e.g.,Allen (2005) Toxicological Sciences 84:110-119; Bhatia (1998)Biotechnology Progress 14:378-387. Adult hepatocytes co-cultured withendothelial cells of the same species showed good maintenance ofliver-specific functions for several weeks in vitro, even though theydid not show significant expansion in cell population. See articles byGuguen-Guilluozo (1983) Experimental Cell Res. 143:47-54; Begue (1983)Biochemical Pharmacology 32:1643-1646. In addition, rat hepatocyteswhich were co-cultured with human fibroblasts and endothelial cells werereported to exhibit stable cytochrome P-450 activity for more than 10days. See, e.g., Kuri-Harcuch and Mendoza-Figueroa (1989)Differentiation 41:148-157; Begue (1983) Biochemical Pharmacology32:1643-1646. Therefore, mixed hepatocyte co-culture systems withnon-liver derived cells may provide microbiological environments similarto those in vivo by optimizing cell-cell interactions. However, thereare still problems about the nature of non-liver derived cells. Theviability and functional activities of co-cultured hepatic primary cellcan be prolonged in vitro, but primary cell proliferation is limited orabsent in these system, which is a critical flaw. Even though severalreports indicate that non-parenchymal liver cells may express functionssimilar to hepatocytes, the nature of non-liver derived cellsco-cultured with liver primary cells has not been establishedunequivocally. See, e.g., Grisham (1980) Annals of the NY Acad. Sci.349:128-137. It is therefore highly desirable to develop culture methodsand culture devices that can allow artificial in vitro (or in vivo)growth of healthy, fully functional and long-lasting liver cells thatcan be transplanted to the patients in need of liver cells.

There is also a critical need for an artificial liver device that canremove toxins and improve immediate and long-term survival of patientssuffering from liver disease. An artificial liver device can be usefulas a temporary artificial liver for patients awaiting a livertransplant, and also provide support for post-transplantation patientsuntil the grafted liver functions adequately to sustain the patient. Oneof the major roadblocks to the development of an effective artificialliver device is the lack of a satisfactory liver cell line that canprovide the functions of a liver.

Yet another benefit of being able to culture healthy liver cells is tomeet the demands for supply of the cells for toxicity testing ofenormous numbers of new or experimental drugs, chemicals, andtherapeutics being developed in the pharmaceutical and chemicalindustry. With the unique toxin-filtering capability of liver cells, anytoxicity of a new drug can be manifested first by the reaction of theliver cells. An array of liver cells can thus be utilized as a fasttesting/screening vehicle to basically simultaneously evaluate thepotential toxicity of many new drugs and compounds.

Two-dimensional and three-dimensionally cultured cells are useful notonly for liver cell related applications, but for producing a number ofother cells in a healthy and accelerated manner. There are needs tosupply or implant various types of cells including bone cells, livercells, kidney cells, blood vessel cells, skin cells, periodontal cells,stem cells, and other human or animal organ cells.

A fast growth and supply of cells especially rare cells, such as stemcell enrichment, can be crucial for many potential therapeuticapplications as well as for enhancing the speed of advances in stem cellscience and technology. In addition, fast detection and diagnosis ofdisease cells or possible bio-terror cells (such as epidemic diseases,anthrax or SARS) from a very small or trace quantity of available cellscan be accomplished if the cell growth speed can be accelerated.

I. Multifunctional Biocompatible Implant and Accelerated Cell GrowthDevices

The invention provides medical devices comprising nano-scaledbiocompatible implantable devices; including compositions (e.g.,articles of manufacture) comprising nano-scaled biocompatibleimplantable devices such as implants (e.g., hip implants, knee implants,elbow implants, Ti rods for broken legs or arms, and the like), andmethods of making and using them. Also provided are compositions andmethods for accelerated cell growth.

SUMMARY

The invention provides compositions and methods for biocompatiblenanostructure materials, devices and fabrication methods. Also providedare compositions and methods which enable maintained, organized and/oraccelerated cell growth, including “mixed cell” growth and/ordifferentiation. There compositions and methods can be useful for avariety of therapeutic, disease diagnosis-prognosis, screening, injuryreconstruction, orthopedic and dental, and cell-tissue supplyapplications.

In one embodiment, compositions and methods are provided forself-organized TiO.sub.2 nanotube arrays grown on titanium metal oralloy substrate to accelerate cell proliferation. In one aspect, thebase material can be pure Ti or can be an alloy based on Ti such asTi—V—Al alloys. Other solid solution-hardened or precipitation-hardenedalloys with increased mechanical strength and durability are alsoprovided.

In another embodiment, compositions and methods are provided for avertically aligned TiO.sub.2 nanotube array adherent on Ti surface whichinduces strong cell adhesion and significantly enhances the formationkinetics of cells and associated bone growth. In one aspect, theTiO.sub.2 nanotubes other biocompatible nanotubes are about from betweenabout 10 to 1000 nm in diameter, about from between about 30 to 300 nmin diameter, or between about 60 to 200 nm in diameter.

In another aspect, the heights of the tubules are determined in part bya desired aspect ratio as relatively short height with an aspect ratioof about less than about 10, or about less than about 5 for ease ofstoring and eventual dispensing of drugs or biological agentsintentionally placed within the tubule cavity. The height is determinedas to reduce a possibility of long tubules breaking off and floatingaround in the human body. In one aspect, the height is from about 40 to800 nm, or about from 100 to 400 nm.

In another aspect, the vertical alignment consists of an open top porethat is necessary for biocompatible implants and other relatedapplications as described herein, as the open top of the nanowire allowsthe penetration of the cells into the nanopore cavity for good adhesion.In one aspect, the configuration of nano-gaps between aligned TiO.sub.2nanotubes is such that nutrients can pass through the bottom and topsurfaces to feed the proliferating cells.

Also provided herein are compositions comprising multifunctional devicesconsisting of vertically aligned nanotubule structures capable ofstoring drugs or other biological agents, including drugs, growthfactors, proteins, enzymes, hormones, antibiotics, antibodies, DNA, andnanoparticles, and methods for making and using them. Other biologicallyactive materials are also provided, such as for example, vitamins andminerals.

The invention provides biocompatible vertically aligned nanotube arraystructures on a biocompatible substrate comprising a laterally separatednanotube arrangement wherein (i) the outer diameter of the nanotube isfrom between about 10 to 1000 nm, from about 30-300 nm, or from about60-200 nm; and, (ii) the inside diameter of the nanotube is at leastabout 20% to 50% of the outer diameter; and (iii) the height of thenanotube is from between about 40 to 800 nm, and from between about 100to 400 nm; and (iv) the aspect ratio is less than about 10, or less thanabout 5; and (v) the vertical alignment angle is within from betweenabout 0 to 45 degrees, and from about 0 to 30 degrees off the verticaldirection; and (vi) the lateral spacing between adjacent nanotubes isfrom between about 2 to 100 nm, and from about 5 to 30 nm; or, anycombination thereof.

In one aspect, the array has a cell-growth accelerating effect, and itfurther comprises cells, e.g., functional cells, such as liver cells,kidney cells, nerve cells, myocytes, stem cells, supportive soft tissuessuch as muscles, skin cells, tendons, fibrous tissues, periodontaltissues, odontoblasts, dentinoblasts, cementoblasts, enameloblasts,odontogenic ectomesenchymal tissue, osteoblasts, osteoclasts,fibroblasts, and other cells and tissues involved in odontogenesis orbone formation, fat, blood vessels, and hard tissues such as bone andteeth, either as a single cell type culture or as a co-culture of atleast two types of cells together, either in vitro or in vivo. In oneaspect, the cell-growth accelerating effect induced by the biomaterialis at least by 25%; 50%; 100%; 200%; 300% or more.

In one aspect, the biocompatible vertically aligned nanotube arraystructure comprises a vertically aligned titanium oxide nanotube arraystructure on a titanium or titanium oxide substrate with a laterallyseparated nanotube arrangement. In one aspect, the sodium titanatenanostructures are superimposed onto the titanium oxide nanotube arraystructure; and in one aspect, hydroxyapatite formation is enhanced uponexposure of the nanotube array structure to simulated or living bodyfluid.

In one aspect, a composition (device) of the invention comprises amatrix material comprising a vertically aligned nanotube array structurecomprising a biocompatible coating materials, e.g., Ti and Ti oxide, orcomprising Zr, Hf, Nb, Ta, Mo, W and/or their alloys and/or oxides ofthese metals or alloys; and in one aspect, comprising a thickness of atleast 1, 2, 3, 4 or 5 or more nm; and in one aspect the coating coverageof at least about 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of thenanotube or nanopore surfaces; and in one aspect the matrix materialcomprises Ti, Zr, Hf Nb, Ta. Mo, W, and/or their oxides, and/or alloysof these metals and oxides, and/or Si, Si oxide, Al, Al oxide, carbon,diamond, noble metals (such as Au, Ag. Pt and their alloys), polymer orplastic materials, or composite metals, ceramics and/or polymers.

In one aspect, the inside pore of the nanotubes comprise at least onebiologically active agent selected from the group consisting ofpharmaceutical compositions, therapeutic drugs, growth factors,proteins, enzymes, hormones, DNA, genes, antibiotics and antibodies. Inone aspect, the inside pore of the nanotubes comprises magneticnanoparticles.

The invention provides accelerated cell growth structures comprising thebiocompatible vertically aligned nanotube array structure of theinvention, and cells, wherein the cells are adherent to the nanotubestructure; and cell growth is accelerated from at least about 25%, 50%,75%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500% or more. Inone aspect, a nutrient fluid is supplied under the growing cells througha gap spacing between the nanotube; and, in one aspect, the nutrientfluid is also supplied from the top of the structure.

The invention provides orthopedic implants comprising the biocompatiblevertically aligned nanotube array structure of the invention, whereinthe surface is modified such that it comprises an adherent titaniumoxide nanotube array; and, in one aspect, upon implantation into ananimal, results in accelerated bone formation.

The invention provides dental implants comprising the biocompatiblevertically aligned nanotube array structure of the invention, whereinthe surface is modified such that it comprises an adherent titaniumoxide nanotube array; and, in one aspect, upon implantation into ananimal, results in accelerated bone formation.

The invention provides multi-functional implant devices comprising thebiocompatible vertically aligned nanotube array structure of theinvention, wherein the vertical pores of the nanotubes contain areservoir of biologically active agents selected from the groupconsisting of pharmaceutical compositions, therapeutic drugs, cancerdrugs, growth factors, proteins, enzymes, hormones. DNA, genes,antibiotics, antibodies, nanoparticles, and, in one aspect, otherbiologically active materials.

The invention provides multi-functional implant devices of theinvention, wherein the device is designed for externally controlledrelease of a colloidal liquid upon application of ultrasonic or magneticstimulation; and, in one aspect, the colloidal liquid comprises abiologically active agent and magnetic nanoparticles; and, in oneaspect, the magnetic nanoparticles are selected from the groupconsisting of biocompatible iron-oxide particles of magnetite(Fe.sub.3O.sub.4) and maghemite (Fe.sub.2O.sub.3, or,.gamma.-Fe.sub.2O.sub.3); and the size of the magnetic nanoparticles isfrom about 5-50 nm in diameter.

The invention provides multi-functional implant devices, wherein a capis deposited at the upper end of the nanotube by oblique incidentsputter deposition on a stationary or a rotating substrate; and, in oneaspect, the cap is narrowed such that the colloidal liquid is retainedin the nanotube before external stimulation for controlled release.

The invention provides methods of externally controlled release of acolloidal liquid into a subject comprising applying external stimulationby alternating current magnetic field to the multi-functional implantdevice of the invention, wherein the magnetic field causes agitation,movement and heat production from the magnetic nanoparticles comprisedin the colloidal liquid resulting in its release from the implantdevice.

The invention provides methods for treating cancer, wherein themulti-functional implant device of the invention is implanted into asubject at the site of cancer; and, in one aspect, external stimulationis applied resulting in the local delivery of anti-cancer drugs andmagnetic hyperthermia treatment.

The invention provides methods of cell proliferation comprising thebiocompatible vertically aligned nanotube array structure of theinvention and adherent cells, wherein upon adhesion the cells areinduced to proliferate; and optionally the cells are grown in vivo, exvivo or in vitro, and after proliferation, the cells are harvested.

The invention provides analytical diagnostic biochips comprising thebiocompatible vertically aligned nanotube array structure of theinvention; wherein the biochip can be used for the rapid diagnosis anddetection of disease cells, cells involved in epidemic diseases orbioterrorism attacks, and cells related to forensic investigations. Inone aspect, of the biocompatible vertically aligned nanotube arraystructures of the invention the nanotube array structure is subdividedalong the X-Y matrix for the rapid detection of disease cells, cellsinvolved in epidemic diseases or bioterrorism attacks, and cells relatedto forensic investigations; and the detection elements comprise themultiplicity of the nanotubes wherein the cells are placed andproliferated; and, in one aspect, the diagnosis and detection techniquesutilized comprise optical detection, chemical detection, biologicaldetection, and magnetic sensor detection.

The invention provides methods for producing biocompatible verticallyaligned nanotube array structure of the invention, comprising: i)vertically aligned, biocompatible titanium oxide nanotubes withdimensions from about 100 nm outer diameter, about 90 nm inner diameter,15 nm wall thickness, and about 250 nm height; and ii) the titaniumoxide nanotube array structure is fabricated by anodization techniqueusing a titanium sheet (optionally 25 nm thick, 99.5% purity) that iselectrochemically processed, for example, in a 0.5% HF solution at 20 Vfor 30 min at room temperature; and iii) to crystallize the depositedamorphous-structure titanium nanotubes into the desired anatase phase,the nanotubes are heat-treated, for example, at about 500.degree. C. forabout 2 hrs. In alternative aspects, the methods for producingbiocompatible vertically aligned nanotube array structure of theinvention comprise: (i) providing a structure comprising verticallyaligned, biocompatible titanium oxide nanotubes having dimensions of atleast about 20, 30, 40, 50, 60, 70, 80, 90 or 100 or more outerdiameter, or in a range from between about 10 to 100 nm outer diameter;and at least about 20, 30, 40, 50, 60, 70, 80, 90 or 100 nm or moreinner diameter, or between about 10 to about 90 nm inner diameter; andat least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nm wallthickness; or between about 10 to 100 nm wall thickness; and/or at leastabout 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225 or250 or more nm in height, or in a range from between about 20 to 300 nmin height; (ii) fabricating a titanium oxide nanotube array structure byanodization technique using a titanium sheet, optionally about at least15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225 ormore nm thick, and optionally at least about 98%, 98.5%, 99%, or 99.5%purity, that optionally is electrochemically processed, and optionallyis electrochemically processed in a 0.5% HF solution at an appliedvoltage of between about 10-30 V for between about 5-200 min, or 20 Vfor 30 min, optionally at room temperature; and (iii) crystallizing thedeposited amorphous-structure titanium nanotubes into an anatase phase,wherein optionally the nanotubes are heat-treated at between about450.degree. C. to 550.degree. C. for between about 0.1-24 hrs, or500.degree. C. for 2 hrs.

Also provided are various methods and uses of the biocompatible nanotubearray devices as described herein, including for example, acceleratingbone growth for orthopedic and dental implant applications;proliferation and harvesting of cells, especially rare cells;therapeutic applications via sustained release of pharmaceuticalcompositions; and rapid diagnosis of diseased cells, or those cellsinvolved in epidemic diseases or bioterrorism attacks.

BRIEF DESCRIPTION OF THE DRAWINGS

The advantages, nature and additional features of the invention willappear more fully upon consideration of the illustrative embodimentsdescribed in the accompanying drawings. In the drawings:

FIG. 1A-B schematically illustrate exemplary devices comprisingself-organized TiO.sub.2 nanotube arrays grown on titanium substrate toaccelerate cell proliferation: FIG. 1A illustrating a vertically alignedTiO.sub.2 nanotube array; FIG. 1B illustrating the array with cell.

FIG. 2A-B illustrate SEM micrographs showing the microstructure of thevertically aligned TiO.sub.2 nanotubes on titanium substrate; FIG. 2Alow magnification, FIG. 2B high magnification.

FIG. 3A-C illustrate micrographs showing structures of the verticallyaligned TiO.sub.2 nanotubes on titanium substrate; FIG. 3A scanningelectron microscope (SEM) micrograph, FIG. 3B longitudinal viewtransmission electron microscope (TEM) micrograph. FIG. 3Ccross-sectional TEM.

FIG. 4 illustrates a SEM micrograph showing the accelerated growth ofhydroxyapatite on the aligned TiO.sub.2 nanotubes about 2-4 times fasterthan flat Ti surface.

FIG. 5A-C illustrate exemplary TiO.sub.2 nanotube array structures withFIG. 5A an illustration of a micrograph of exemplary nano-inspiredsodium titanate nanofiber structure on the ends of TiO.sub.2 nanotubes;FIG. 5B a schematic illustration of an exemplary sodium titanatenanofiber structure; and FIG. 5C an illustration of a micrograph of anexemplary nanoscale hydroxyapatite phase rapidly formed on the NaOHtreated TiO.sub.2 nanotubes at a speed of about seven times faster thanwithout the NaOH treatment.

FIG. 6A-B illustrate comparative SEM micrographs showing the acceleratedgrowth of osteoblast cells on the vertically aligned TiO.sub.2 nanotubesof the invention (on anatase TiO.sub.2 nanotubes at 2 hours), FIG. 6B,as compared to the flat Ti surface (pure Ti, 12 hour growth), FIG. 6A.

FIG. 7A-B illustrate micrographs showing the growth and adhesion ofosteoblast cell on and into vertically nanoporous TiO.sub.2 nanotubes ofthe invention; FIG. 7A low magnification, FIG. 7B higher magnification.

FIG. 8A-C illustrate back scattered electron SEM images of osteoblastcells on: FIG. 8A only Ti, FIG. 8B as-deposited (amorphous) alignedTiO.sub.2 nanotubes, and FIG. 8C annealed, anatase TiO.sub.2 nanotubesof the invention.

FIG. 9 illustrates a plot of the counted number of adhered cells (persquare centimeter) as a function of incubation period in hours on thesurface of Ti only, amorphous TiO.sub.2 nanotubes, and anatase TiO.sub.2nanotubes of the invention.

FIG. 10A-B illustrate comparative pictures of stem cell adhesion andgrowth shown as back scattered electron SEM images; FIG. 10A on flat Tisurface, FIG. 10B on an exemplary anatase-phase vertically alignedTiO.sub.2 nanotube array of the invention.

FIG. 11A-C illustrate various exemplary orthopedic body implants of theinvention comprising TiO.sub.2 nanotubes or associated variations of theinvention; FIG. 11A, illustrating implants as orthopaedic and dentalimplants, including dental and periodontal implants, elbow implants, hipimplants, knee implants, leg implants; FIG. 11B illustrating implants asimplanted cells or organs, e.g., an artificial liver device; FIG. 11Cillustrating implants as drug delivery devices for, e.g., stents,therapeutic devices, e.g., with insulin, or for cancer.

FIG. 12A-D illustrate examples of TiO.sub.2 nanotube-based implants ofthe invention containing slow-releasing biological agents stored in thevertically aligned nanotube pores, and the process of cell growth: FIG.12A, with TiO.sub.2 nanotubes (on a Ti substrate); FIG. 12B, withbiological additives; FIG. 12C, with cells; FIG. 12D, with growing cellsadherent to the TiO.sub.2 nanotubes.

FIG. 13A-B illustrate examples of TiO.sub.2 nanotube-based implants ofthe invention comprising: FIG. 13A nanotubes with therapeutic agentsplus magnetic nanoparticles stored in the vertically aligned nanotubepores; and FIG. 13B the process of drug release via magnetic particlemovement or heating by the onset of applied magnetic field.

FIG. 14A-B illustrate an exemplary implant device of the invention inwhich the top open ends of the vertically aligned TiO.sub.2 nanotubeswhich are intentionally made to be narrower in passage diameter afterthe magnetic nanoparticles are incorporated, FIG. 14A; and an exemplaryimplant device with therapeutic medicine, FIG. 14B.

FIG. 15 is a schematic illustration of a patient treated by a controlleddrug release implant device of the invention actuated by externalstimuli—in this example an electromagnet for generating an AC magneticfield.

FIG. 16A-B are schematic illustrations of an exemplarycell-proliferation device of the invention based on TiO.sub.2 nanotubes;showing cells proliferating on the TiO.sub.2 nanotubes. FIG. 16A; and aschematic of cell harvesting by trypsinization (followed bycentrifugation), FIG. 16B.

FIG. 17 is a schematic illustration of an exemplary X-Y matrixsubdivided array of TiO.sub.2 nanotube array structure of the inventionfor accelerated cell, bacterial or virus growth on a diagnostic biochip;detection elements comprising TiO.sub.2 nanotube array structures upon abiochip substrate are illustrated.

FIG. 18: FIG. 18A-C is a schematic illustration of an exemplary cellanalysis device which comprises TiO.sub.2 nanotube arrays capable ofaccelerating cell proliferation to enhance cell-based assays; FIG. 18A,illustrates optical detection by, e.g., microscope, fluorescentmicroscope, or CCD camera sensing of fluorescent or quantum dot taggedcells; FIG. 18B, illustrates chemical or biological detection, e.g.,based on signature reactions; FIG. 18C, illustrates magnetic sensordetection, e.g., by using magnetically targeted antibody.

FIG. 19A to 19C: schematically illustrate exemplary processing steps forguided anodization of Ti using pre-fabricated nano spot array of theinvention for controlled TiO.sub.2 nanotube geometry; 19A, illustratespreparing Ti metal sheet, foil or film; 19B, illustrates use oflithography or self-assembly process to make nano spot array at desiredperiodicity; 19C, illustrates guided anodization to fabricate aTiO.sub.2 nanotube array with predetermined size and periodicity.

FIG. 20A to 20C: schematically illustrate an exemplary lithographicalfabrication of gapped nanotube array of the invention followed bycoating with a biocompatible layer such as Ti or TiO.sub.2; FIG. 20A,illustrates preparing Si or any other directionally-etchable material;FIG. 20B, illustrates an exemplary lithographically patterned nanotubearray; FIG. 20C, illustrates the deposit of Ti or TiO.sub.2 coating tocover the nanotube surface.

FIG. 21A to 21E: schematically illustrate an alternative exemplarymethod of creating biocompatible nanotube array of the inventioncomprising steps of lithographical fabrication of nanocavity, insidewall deposition, matrix etching for nanotube array, followed by coatingwith a biocompatible layer such as Ti or TiO.sub.2; FIG. 21A,illustrates preparing an Al or Si layer (bulk or deposited film); FIG.21B, illustrates the step of lithographically patterning a hole array(round or square cavities); FIG. 21C, illustrates the step of depositingbiocompatible metal or ceramic cylinder (e.g., Au. Pd, carbon); FIG.21D, illustrates the step of etching off the matrix to produce open-porecylinder array; FIG. 21E, illustrates the step of depositing Ti orTiO.sub.2 coating to cover the surface.

FIG. 22A to 22C: schematically illustrate an exemplary process ofpreparing channeled array of the invention, comprising parallel-alignednanocavities comprising a surface coating of biocompatible layer such asTi or TiO.sub.2; FIG. 22A illustrates an exemplary patterned nano-cavityarray, made e.g., by lithography or self-assembly process, on Si, anymetallic, ceramic or plastic substrate; FIG. 22B illustrates the step ofcoating the surface with biocompatible Ti or TiO.sub.2 film; FIG. 22Cillustrates the step of providing sideway linear or x-y crossing groovepaths to allow fluid flow.

FIG. 23A to 23D: schematically illustrate an accelerated 3-dimensionalcell growth using exemplary structures of the invention comprisingretractable or permanently retained sheets with parallel-aligned andgapped nanotube array on their surfaces; FIG. 23A illustrates anexemplary device of the invention comprising parallel-configured,retractable Ti wires or ribbons with a TiO.sub.2 nanotube surface; FIG.23B illustrates the step of accelerated cell growth on the parallel Tiwires or ribbons (e.g., for liver, blood vessel, bone cells, etc.); FIG.23C illustrates the step of optionally pulling out titanium oxidenanotubes; FIG. 23D illustrates the final result of practicing thisexemplary device and method of the invention—a cultured 3-dimensionalcell system.

FIG. 24 schematically illustrates an exemplary bio-chip device fortoxicity testing of drugs or chemicals, with the device comprising anarray of cultured liver cells grown in a rapidly and healthy manner onan parallel-aligned and laterally spaced nanotubes coated device of theinvention, with biocompatible surface layer.

FIG. 25A-B show comparative backscattered electron SEM images of livercell growth on FIG. 25A sand blasted, roughened Ti; FIG. 25B exemplaryTiO.sub.2 nanotube array surface of the invention.

FIG. 26A-B schematically illustrate cell growth on exemplary TiO.sub.2nanotubes of the invention in different positions; FIG. 26A, verticallypositioned; FIG. 26B, horizontally upright positioned; FIG. 26C,horizontally inverted positioned.

FIG. 27A-B schematically illustrate accelerated growth of 3-dimensional,tube-shaped cells such as blood vessel cells, as in FIG. 27A, orenzyme/hormone secretion tubes, cultured using an exemplary retractableTi wire array substrate of the invention, as in FIG. 27B, with each wirehaving a TiO.sub.2 nanotube arrayed surface (a pull out titanium wirearray substrate with each wire having titanium oxide surface).

FIG. 28A-C schematically illustrate the exemplary 3-dimensional cellgrowth process of culturing at least two types of cells together on a Tiwire array of the invention, comprising TiO.sub.2 nanotube surfaceaccording to the invention; FIG. 28A illustrates the growth of nonparenchymal stromal cells (e.g., blood vessel cells) on Ti; FIG. 28Billustrates the growth of parenchymal cells (e.g., liver cells); FIG.28C illustrates the step of removing the Ti wire array by it pulling outfrom the growing cells.

FIG. 29A-D schematically illustrate accelerated culture of 3-D cellswith permanently retained Ti wires, e.g., bent wires on Ti sheetsubstrate, as illustrated in FIG. 29A; coiled Ti wire array, asillustrated in FIG. 29B; networks or sponges having TiO.sub.2 nanotubearrayed surfaces, e.g., Ti wire mesh, as illustrated in FIG. 29C; and,ribbons, e.g., curved Ti ribbons, as illustrated in FIG. 29D.

FIGS. 30A-E schematically illustrate exemplary configurations of adimensionally controlled, lock-in structure with the size or diameter ofthe entrance of the pores made smaller by oblique incident deposition ofbiocompatible materials such as Ti or TiO.sub.2; FIG. 30A illustrates anexemplary nano- or micro-pore array comprising implant material (e.g.,Ti, TiO.sub.2, Zr, ZrO.sub.2, etc., Si, SiO.sub.2, polymers, metalsceramics, composites); FIG. 30B illustrates an exemplary nano- ormicro-pore array comprising oblique deposition of Ti, TiO.sub.2, Zr,ZrO.sub.2, etc.; FIG. 30C illustrates an exemplary nano- or micro-porearray comprising additional diameter-reducing deposit (e.g., of Ti.TiO.sub.2, Zr, ZrO.sub.2, etc.) on pore entrance by substrate rotation;FIG. 30D illustrates an exemplary nano- or micro-pore array comprising adiameter-reducing deposit (e.g., of Ti, TiO.sub.2, Zr, ZrO.sub.2, etc.)on the nanotube and gap entrance; FIG. 30E illustrates an exemplarynano- or micro-pore array comprising diameter-reducing deposit (e.g., ofTi. TiO.sub.2, Zr, ZrO.sub.2, etc.) on entrance to random pores.

FIGS. 31A-D schematically illustrate alternative types of lock-innanostructures of the invention, e.g., for enhanced mechanical stabilityof cells or grown hard structures, e.g., grown bones or teeth: FIG. 31Aillustrates random-diametered re-entrant oval or circular nanopores(e.g., with an exemplary implant or bio-substrate of Ti, TiO.sub.2, Zr,ZrO.sub.2, Zr, Hf, Nb, Ta, Mo, W and/or their oxides, and/or alloys ofthese metals or oxides); FIG. 31B illustrates rectangular cavity withcorrugating walls (an exemplary implant comprising corrugated nanoporearray); FIG. 31C illustrates re-entrant triangular cross-sectioned pores(illustrating an exemplary implant comprising a pore with a gradientdiameter having an expanding dimension from the pore entrance); FIG. 31Dillustrates exemplary nanotubes with corrugating walls (an exemplaryimplant comprising nanotube with corrugated walls).

FIGS. 32A-B schematically illustrate titanium nanotubes of the inventionformed by electrolytic anodization, showing the limited range ofnanotube diameter control available by voltage control, and thedependence of TiO.sub.2 nanotube diameter on anodization voltageapplied; FIG. 32A illustrates an exemplary anodization processing at 15V for 30 min in 0.5% HF at room temperature; FIG. 32B illustrates anexemplary anodization comprising processing at 20 V for 30 min in 0.5%HF at room temperature.

FIG. 33 and FIG. 34 schematically illustrate an exemplarymicro-nano-dual pore structure of the invention (a micro-nano-dual porestructure of anisotropically ion etched microcavities andanodization-induced surface TiO.sub.2 nano tubes) comprising: FIG. 33,anisotropically ion etched microcavities and anodization-induced surfaceTiO.sub.2 nanotubes; and FIG. 34, isotropically etched microcavities andanodization-induced surface TiO.sub.2 nanotubes.

FIGS. 35A-C schematically illustrate higher magnification illustrationsof FIG. 33 and FIG. 34 structures showing the details of the titaniumoxide nanotubes formed on the top and pore surfaces; FIG. 35A, anexemplary box-shaped pore; FIG. 35B, an exemplary narrow-orificed shapedpore; FIG. 35C, an exemplary round-shaped pore.

FIG. 36 schematically illustrates an exemplary procedure and structurefor guided synthesis of enlarged-diameter nanotubes and nanopores ofTiO.sub.2 using pre-patterning of craters on Ti or TiO.sub.2 surface (anexemplary guided synthesis protocol for fabrication of larger diameternanotubes and nanopores with e.g., greater than 200 nm, 300 nm, 400 nm,or more in diameter); including illustration of pre-patterned craters inTi make, e.g., by e-beam litho, photolitho, nano imprint litho, etc.,and chemical etch or anodizing.

FIGS. 37A-C schematically illustrate an exemplary nano-imprintinglithography process comprising use of nano imprinting to pre-patterncraters on Ti or TiO.sub.2 surface for guided synthesis of largerdiameter TiO.sub.2 nanotubes and nanopores; FIG. 37A illustrates anexemplary imprinting process; FIG. 37B illustrates an exemplary patternformation process; FIG. 37C illustrates an exemplary pre-pattern etchand anodization process.

FIGS. 38A-B schematically illustrate exemplary processes of guided etchnano patterning on non-flat surfaces using: FIG. 38A, on, e.g., acylinder or random shape substrate and/or implant, using a conformableor stretchable elastomeric mask sheet (using conformable and/orstretchable mask sheets for guided patterning on non-flat surfaces);FIG. 38B elastomeric roll stamping (elastomeric nano-implant stamp forroll stamping of surface patterns for local etching and guidedpatterning on cylindrical/round substrate or implant).

FIGS. 39A-F schematically illustrate exemplary uniform nanopore ornanotube arrays of the invention on non-flat surface by guided etchingusing a vertically two-phase decomposed coating: FIG. 39A, illustrates astarting cylinder or random shape substrate/implant (e.g., Ti wire)material; FIG. 39B, illustrates the step of coating of textured material(e.g., co-sputtered layer, decomposable diblock copolymer, spinodallydecomposing alloy, etc.) on the starting cylinder or random shapesubstrate/implant; FIG. 39C, illustrates the resultant nanopored coatingafter preferential etching away of one phase; FIG. 39D, illustrates thestep of etching through the pore for formation of guiding craters; FIG.39E, illustrates the step of removing the coating; FIG. 39F, illustratesthe optional step of additional etching or anodization to produce deepernanopores or nanotubes on the implant surface.

FIGS. 40A-E schematically illustrate exemplary size-controlled, uniformnanopore or nanotube array on various surfaces by guided etching using avertically two-phase decomposed coating of periodically or spinodallydecomposing alloy, which optionally can comprise biological agents orfunctional nanoparticles stored in the pores on various shapednano-structure surfaces, such as magnetic particles, metals or SPR(surface plasmon resonance) particles, quantum dots, fluorescenceparticles, bio-conjugated particles, for (accelerated) cell/bone growth,protein harvest, delivery of drugs, genes, chemicals, therapeutics, etc:FIG. 40A, illustrates an exemplary nanopore or nanotube array on a flatsurface; FIG. 40B, illustrates an exemplary array as a coarse-patternedsurface; FIG. 40C, illustrates an exemplary array as a parallel Ti sheetor wire array; FIG. 40D, illustrates an exemplary array as a wire mesh,bundle or foam (e.g., made of Ti); FIG. 40E, illustrates an exemplaryarray as a re-entrant cavity surface (e.g., Ti or Ti-coated Si).

FIGS. 41A-D schematically illustrate exemplary bone or cell lockingnanopore and/or nanotube array with biological agents inserted foraccelerated bone or cell growth, protein or hormone harvest, drugdelivery, and therapeutics: FIG. 41A, illustrates an exemplary nano porearray with reduced entrance dimension (Ti, TiO.sub.2, etc) and a trappedbiological agent (e.g., collagen, growth factor, magnetic particles,DNAs, antibiotics, therapeutic drugs, etc.); FIG. 41B illustrates anexemplary nanopore array comprising various bone-locking or cell-lockingshapes of nanopores for convenient storage of biological agents, e.g.,collagen, etc.; FIG. 41C illustrates an exemplary nanopore arraycomprising nanotube (e.g., TiO.sub.2 or coated with TiO.sub.2) havingdiameter-reducing deposits on the nanotube and/or gap entrances; FIG.41D illustrates collagen, etc. inside bone-locking or cell-lockingrandom shapes of exemplary nanopores.

FIGS. 42A-D schematically illustrate exemplary arrays comprising cellsor bone growing and locked-in on a re-entrant shaped nano-structuresurface: FIG. 42A, schematically illustrates an exemplary nanopore arraywith reduced entrance dimension, optionally containing biologicalagents, and cells (or bone) growing and locked in on the re-entrantshaped nano-structure surface; FIG. 42B, schematically illustrates anexemplary nanopore array comprising various bone-locking or cell-lockingshapes of nanopores (with optional biological agents); FIG. 42C,schematically illustrates an exemplary nanotube (e.g., TiO.sub.2 orcoated with TiO.sub.2) comprising a diameter-reducing deposit onnanotube and gap entrance; FIG. 42D, schematically illustratesbone-locking or cell-locking random shapes of exemplary nanopores (withoptional biological agents).

FIGS. 43A-D schematically illustrate exemplary nanopore or nanotubearray with functional nanoparticles stored in the pore on various shapednano-structured surfaces for accelerated cell or bone growth, proteinharvest, drug delivery, and therapeutics: FIG. 43A, schematicallyillustrates an exemplar) nanopore array with reduced entrance dimension(Ti, TiO.sub.2, etc), comprising trapped functional nanoparticles(magnetic particles, novel metal or SPR particles, quantum dots,fluorescence particles, bio-conjugated particles for delivery of drugs,genes, chemicals, etc.); FIG. 43B, schematically illustrates exemplarybone-locking or cell-locking shapes of nanopores of the invention forconvenient storage of biological agents, magnetic particles, etc.; FIG.43C schematically illustrates an exemplary nanotube (e.g., TiO.sub.2 orcoated with TiO.sub.2) comprising a diameter-reducing deposit onnanotube and gap entrance; FIG. 43D, schematically illustrates exemplarypores comprising inserted nano- or microparticles (e.g., magneticparticles, quantum dots, fluorescence particles, bio-conjugatedparticles for delivery of drugs, genes, chemicals, etc.) insidebone-locking or cell-locking random shapes of nanopores.

FIG. 44 schematically illustrates an exemplary cell growth devicecomprising the dimension-controlled biomaterials as an array on abiochip (for use, e.g., as an array for drug toxicity testing), and anarray of size-controlled (and varied sized) nanopores and/or nanotubes.

FIG. 45 schematically illustrates an exemplary biochip device, e.g., fortoxicity testing of drugs or chemicals, with the device comprising anarray of cultured cells. e.g., liver cells, grown ondimension-controlled biomaterials, wherein in one aspect the arraycomprises cultured liver cells optionally co-cultured with other typesof cells.

FIGS. 46C-E schematically illustrate exemplary structures of theinvention comprising configurations of exemplary size-randomizednanopore structures of biocompatible materials (e.g., for acceleratedcell/bone growth, e.g., multi-cell-type co-culture, protein harvest,drug delivery, therapeutics, etc.), comprising Ti or TiO.sub.2, with thesize or diameter of the entrance of the pores having a varieddistribution of dimensions, as illustrated in FIG. 46C-E, as compared tothe uniform sized nanopores FIGS. 46A and 46B; FIG. 46A, top view of anexemplary equal-diameter nanopore array; FIG. 46B, side view ofexemplary equal-diameter pores; FIG. 46C, top view of random-sizedexemplary nanopores; FIG. 46D, side view of exemplary random-sizedpores; FIG. 46E, nanopore structure with large and small cells.

FIGS. 47A-F schematically illustrate exemplary structures of theinvention comprising size-randomized pore formation on flat surface,e.g., illustrate an exemplary process for making a randomized poreformation on, e.g., a Ti wire, mesh, or sheet surface usingsemi-wettable or island-forming coating (e.g., for accelerated cell/bonegrowth especially multi-cell-type co-culture): FIG. 47A illustrates astarting material comprising, e.g., a cylinder or random shapesubstrate/implant (e.g., Ti wire); FIG. 47B illustrates application of asemi-wettable coating, which can be broken up or balled up into islands(e.g., a spray- or dip-coated polymer which balls up on drying, or asputtered or evaporated metal film which balls up into islands onheating); FIG. 47C illustrates formation of balled up islands withrandom distribution of sizes; FIG. 47D illustrates the step of chemicalor electrochemical oxidation into TiO.sub.2 surface oxide except theisland regions; FIG. 47E illustrates the step of dissolving away theislands to form random sized craters; FIG. 47F illustrates the step ofchemical etching or anodization using the craters as the preferredreaction sites for deeper nanopores on the implant surface.

FIGS. 48A-E schematically illustrate exemplary structures of theinvention comprising size-randomized pore formation on flat or non-flatTiO.sub.2 covered surface such as a Ti plate, sheet, wire, mesh, or foamby using semi-wettable or island-forming coating as a local mask: 48A,illustrates a starting material comprising, e.g., a cylinder or randomshape substrate/implant pre-oxidized Ti (e.g., Ti wire with TiO.sub.2surface coating); 48B illustrates application of a semi-wettable coatingwhich can be broken up or balled up into islands (e.g., a spray- ordip-coated polymer which balls up on drying, or a sputtered orevaporated metal film which balls up into islands on heating); 48Cillustrates formation of balled up islands with random distribution ofsizes; 48D illustrates the step of chemical or electrochemical etchingof TiO.sub.2 surface oxide except the island regions to form craters;48E illustrates the step of chemical etching or anodization using thecraters as the preferred reaction sites for deeper nanopores ornanotubes on the implant surface.

FIGS. 49A-D schematically illustrate an exemplary process, a randomizedpore formation on non-flat substrates such as Ti wire, rod, mesh surfaceusing nanoparticle-containing coating, this process being an alternativemethod of using a polymer or an aqueous solution containingnanoparticles of coating material (polymer, metal or salt) for formationof island masks; FIG. 49A, illustrates a starting material comprising,e.g., a cylinder or random shape substrate/implant pre-oxidized Ti(e.g., Ti wire with TiO.sub.2 surface coating); FIG. 49B, illustratesapplication of a coating containing nanoparticles of polymer, metal orsalt; FIG. 49C, illustrates the step of drying or heating to haveisolated islands of nanoparticles as mask islands; FIG. 49D, illustratesthe step of chemical etching or anodization through mask islands, orformation of Ti-oxide coating except the mask islands followed bychemical etching or anodization.

FIGS. 50A-F schematically illustrate exemplary structures of theinvention comprising randomized pore sizes and shapes fabricated by thinfilm deposition. FIGS. 50A-B, illustrate as-deposited porous structure;FIGS. 50C-D, F, illustrate rough surfaced thin film followed byanodization; FIG. 50A illustrates an exemplary array surface comprisinga porous thin film made by sputtering or evaporation (e.g., Ti); FIG.50B illustrates an exemplary array surface oxidized by anodization orO.sub.2 annealing; FIG. 50C illustrates an exemplary array surfacecomprising a rough-surfaced thin film made by sputtering or evaporation(e.g., Ti, TiO.sub.2, Zr, Ta, Hf. Si, SiO.sub.2); FIG. 50D illustratesan exemplary array surface comprising a porous pillar or nanotubestructure by anodization (e.g., into TiO.sub.2); FIG. 50E illustratesthe exemplary 50A or 50B with large and small cells; FIG. 50Fillustrates the exemplary 50C or 50D with large and small cells.

FIGS. 51A-F schematically illustrate exemplary structures of theinvention comprising randomized pore size and shape fabricated by thinfilm deposition, with a biological agent inserted into the nanopores:FIGS. 51 A-C, illustrate as-deposited porous structure; FIG. 51D-(f)illustrate rough surfaced thin film followed by anodization; FIG. 51Aillustrates an exemplary array surface comprising a porous thin filmmade by sputtering or evaporation (e.g., Ti); FIG. 51B illustrates anexemplary array surface oxidized by anodization or O.sub.2 annealing,with another composition, e.g., a growth factor, a collagen; FIG. 51Cillustrates the exemplary 51A or 51B with large and small cells; FIG.51D illustrates an exemplary array surface comprising a rough-surfacedthin film made by sputtering or evaporation (e.g., Ti, TiO.sub.2, Zr,Ta. Hf, Si, SiO.sub.2); FIG. 51E illustrates an exemplary array surfacecomprising a porous pillar or nanotube structure by anodization (e.g.,into TiO.sub.2), with another composition, e.g., a growth factor, acollagen; FIG. 51F illustrates the exemplary 50D or 50E with large andsmall cells.

FIGS. 52A-C schematically illustrate exemplary structures of theinvention comprising porous TiO.sub.2 structure made by evaporation, orsputtering process as illustrated in FIG. 52A; or as sputtered porousstructure; or as illustrated in FIG. 52B with a collagen type biologicalagent inserted into the nanopores; or as illustrated in FIG. 52C withfunctional nanoparticles (such as magnetic or other movable functionalparticles, including, e.g., drugs, nucleic acids, growth factors,hormones, etc., for local heating, local magnetic field generation,generating electrical impulses, drug delivery, etc) inserted into thenanopores.

FIGS. 53A-D schematically illustrate exemplary structures of theinvention comprising lithographically created random-sized pores ornanotubes according to the invention (for accelerated cell/bone growth,e.g., multi-cell-type co-culture, protein harvest, drug delivery,therapeutics, etc.): FIG. 53A illustrates use of lithography through arandomly shaped mask (e.g., by photo-, electron-, ion-,nanoimprint-lithography, or laser speckle interference pattern) togenerate random-sized pores or nanotubes; FIG. 53B illustrates theresultant etched random-size pore or pillar pattern (with optionalcoating with TiO.sub.2, etc.);

FIG. 53C illustrates biological agents inserted into the nanopores, witha cultured cell, FIG. 53D illustrates functional nanoparticles addedinto the nanopores, wherein the pores can also contains (or thenanopores can also comprise) a biological agent, e.g., a drug, nucleicacid, a growth factor, hormones, magnetic or other movable functionalparticles, or bio-conjugated particles, etc., for, e.g., accelerated ordelayed biological reaction, for local heating, local magnetic field,electrical impulses, controlled drug delivery, therapeutics, etc.

FIGS. 54A-D schematically illustrate alternative types of lock-innanostructures of the invention for enhanced mechanical stability oftissues, e.g., grown bones, each nanostructure, e.g., nanopore ornanotube, also comprising a biological agent, e.g., a drug, nucleicacid, a growth factor, hormones, magnetic or other movable functionalparticles, or bio-conjugated particles, etc., for, e.g., accelerated ordelayed biological reaction, for local heating, local magnetic field,electrical impulses, controlled drug delivery, therapeutics, etc., and acell adherent to the pore/tubule opening: FIG. 54A illustratesrandom-diametered re-entrant oval or circular nanopores (e.g., with anexemplary implant or bio-substrate of Ti, TiO.sub.2, Zr. ZrO.sub.2, Zr,Hf, Nb, Ta, Mo, W and/or their oxides, and/or alloys of these metals oroxides); FIG. 54B rectangular cavity with corrugating walls (anexemplary implant comprising corrugated nanopore array); FIG. 54Cre-entrant triangular cross-sectioned pores (illustrating an exemplaryimplant comprising a pore with a gradient diameter having an expandingdimension from the pore entrance); FIG. 54D nanotubes with corrugatingwalls (an exemplary implant comprising nanotube with corrugated walls).

FIGS. 55A-E schematically illustrate the growth/maintenance of differentsize/shape cells grown on exemplary size-randomized nanopore or nanotubearrays of the invention comprising various shaped surfaces by guidedetching using semi-wettable or island-forming coating, or porous thinfilm deposition: FIG. 55A, illustrates an exemplary nanopore or nanotubearray on a flat surface, with cells; FIG. 55B, illustrates an exemplaryarray as a coarse-patterned surface, with cells; FIG. 55C, illustratesan exemplary array as a parallel Ti sheet or wire array, withgrowth/maintenance of cells, tissues and/or organs; FIG. 55D,illustrates an exemplary array as a wire mesh, bundle or foam (e.g.,made of Ti), with growth/maintenance of cells, tissues and/or organs;FIG. 55E, illustrates an exemplary array as a re-entrant cavity surface(e.g., Ti or Ti-coated Si).

FIGS. 56A-C schematically illustrate exemplary processes of guided etchnano-patterning of diameter-randomized nanopores or nanotubes; FIG. 56A,illustrates guided etch nano-patterning on non-flat surfaces usingconformable or stretchable elastomeric mask sheet on a cylinder orrandom shape substrate/implant; FIG. 56B, illustrates guided etchnano-patterning using elastomeric roll stamping (elastomericnano-implant stamp for roll stamping of surface patterns for localetching and guided patterning); FIG. 56C, illustrates guided etchnano-patterning using elastomeric flat stamping on large area surfaces(an exemplary process for patterning on a large-area, flat surface usingelastomeric nano stamping; in alternative aspects, a Reactive IonEtching (RIE) approach or direct stamping of island etch mask (or mirrorimage mask) are used).

FIGS. 57A-B schematically illustrate FIG. 57A cell proliferation onsize-randomized nanopores or nanotubes; and FIG. 57B an exemplary methodof harvesting the cells cultured using trypsinization and/orcentrifugation.

FIG. 58 schematically illustrates an exemplary biochip device comprisingsize-randomized nanopores or nanotubes, e.g., for toxicity testing ofdrugs or chemicals, with the device comprising an array of culturedcells, e.g., liver cells grown on dimension-controlled biomaterials,wherein in one aspect the array comprises cultured liver cellsoptionally co-cultured with other types of cells.

It is to be understood that the drawings are for purposes ofillustrating the concepts of the invention and are not to scale.

DETAILED DESCRIPTION

The invention provides compositions comprising multifunctionalbiocompatible implants and devices that accelerate cell growthcomprising (or consisting of) biocompatible aligned nanotubulestructures and methods for fabricating such devices, and methods formaking and using them.

The invention provides compositions comprising vertically alignedTiO.sub.2 nanotube arrays adherent on titanium surfaces, see, e.g.,FIGS. 1, 2 and 3. Such nanotube arrays are capable of inducing strongcell adhesion and inducing rapid proliferation of cells, such as thoseinvolved in bone formation. The configuration of nano-gaps between thealigned TiO.sub.2 nanotubes is such that nutrients can pass between thebottom as well as the top surface in order to feed the proliferatingadherent cells. Adherent cells are generally healthy and fast growing,while the non-adherent cells often exhibit reduced or minimal growth.

Biocompatible implants consisting of TiO.sub.2 nanotubes are providedthat have use in osteogenic and dental applications. Also provided aremultifunctional TiO.sub.2 nanotubes devices capable of storingpharmaceutical compositions and biological agents. Examples includedrugs, growth factors, hormones, proteins, enzymes, antibiotics,antibodies, DNA, nanoparticles, vitamins and minerals. The biocompatibleTiO.sub.2 nanotubes as described herein, are useful in a variety ofapplications including accelerating bone growth for orthopedic anddental repair; in vivo and in vitro accelerated growth of cellsincluding functional cells (such as liver cells, kidney cells, nervecells, myocytes, stem cells) or supportive tissues (soft tissues such asmuscles, tendons, fibrous tissues, periodontal tissues, fat, bloodvessels, or hard tissues such as bone and teeth), proliferation and/orharvesting of cells to be supplied for therapeutics and laboratoryexperiments, particularly rare cell types such as stem cells or diseasecells; therapeutic applications for local sustained drug release; andrapid diagnosis of cell-based conditions, toxicities and/or diseasesinvolved in, for example, infections, epidemics and/or biologicalwarfare agent or toxin exposures.

EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Bone- and Cell-Growth Promoting Nanostructures

An example of an exemplary bone- and cell-growth promoting nanostructureof the invention is shown in FIGS. 2 and 3. These exemplary structuresof the invention are vertically aligned, biocompatible TiO.sub.2 with atypical dimension of the hollow nanotubes as shown as beingapproximately (about) 100 nm outer diameter and approximately 70 nminner diameter, with approximately 15 nm in wall thickness, andapproximately 250 nm in height.

The exemplary TiO.sub.2 nanotube array structure shown in FIGS. 1-3 wasfabricated by an exemplary anodization technique using a Ti sheet (0.25mm thick, 99.5% purity) which is electrochemically processed in a 0.5%HF solution at 20 V for 30 min at room temperature. A platinum electrode(thickness: 0.1 mm, purity: 99.99%) was used as the cathode. Tocrystallize the deposited amorphous-structured TiO.sub.2 nanotubes intothe desired anatase phase, the specimens were heat-treated at500.degree. C. for 2 hrs. In one aspect, the amorphous TiO.sub.2nanotubes are crystallized to anatase phase by heat treatment, becausean amorphous TiO.sub.2 phase tends to be more susceptible to breakage byexternal stresses as compared to a crystalline phase.

For evaluation of bone growth on bioactive surface in terms ofhydroxyapatite (HAp) formation, the TiO.sub.2 nanotube specimens of FIG.2 were soaked for 1, 2, 3 and 5 days, in 20 mL of a simulated body fluid(SBF) solution at 36.5.degree. C., which contained ion concentrationsnearly equal to those of human blood plasma with respect to Na.sup.+,K.sup.+, Ca.sup.2+, Mg.sup.2+, Cl.sup.−, HCO.sub.3.sup.−,HPO.sub.4.sup.2−, and SO.sub.4.sup.2− concentrations. After apredetermined soaking time, the specimens were removed from the SBFsolution, gently rinsed with distilled water, and then dried at60.degree. C. for 24 hrs.

Another important factor for healthy cell growth is a continuous supplyof nutrients (e.g. proteins, mineral ions, fluid, etc.) to the cellthrough the flow of body fluid. The gap (i.e. spacing) between adjacentTiO.sub.2 nanotubules in FIGS. 1-3 serves such a function by allowingthe body fluid to continuously pass through and thereby supply nutrientsto the bottom side of the growing cells. The desired gap between thenanotubules is in the range of about 2-100 nm, or about 5-30 nm. Toosmall a gap reduces the effectiveness of nutrient body fluid flow whiletoo large a gap can pose a danger of reduced mechanical stability in theevent of vertical or lateral stress or pressure. A transmission electronmicroscope (TEM) photograph shown for an exemplary TiO.sub.2 nanotubulearray structure, FIGS. 3B and 3C, gives an average of approximately 15nm spacing between the nanotubes. The SEM micrograph in FIG. 4 shows theaccelerated growth of hydroxyapatite on the aligned TiO.sub.2 nanotubeswhich occurred, at least about 2-4 times faster than on flat Ti surface.

Example 2 Nanofiber-Like or Nanoribbon-Like Structures

On exposure of the TiO.sub.2 nanotubes to a 5 mole NaOH solution atapproximately 60.degree. C. for 60 minutes, it has been found that anadditional, extremely fine, and predominantly nanofiber-like ornanoribbon-like structure of sodium titanate compound is introduced onthe very top of the TiO.sub.2 nanotubes as shown in FIG. 5A. In thisexample, preferential occurrence of nanofibers at the top of nanotubesis presumably because of the nanotube contact with NaOH solution aboveand also possibly due to the surface-tension-related difficulty of NaOHsolution getting into nanopores within and in-between TiO.sub.2nanotubes, as illustrated in FIG. 5B. Compositional analysis by EDXA(energy dispersive x-ray analysis) in SEM confirms the presence of Na,Ti and O after the exposure of TiO.sub.2 nanotubes to NaOH. The sodiumtitanate so introduced exhibits an extremely fine-scale nanofiberconfiguration with a dimension of approximately 8 nm in average diameterand approximately 50-100 nm long.

The growth of even finer scale structure from a given nanostructure asdemonstrated in FIG. 5 can be of significant interest for basicmaterials development for nanotechnology, since such a concept can beutilized as one of the novel and efficient ways of creating extremelyfine nanostructures in many different materials. It is believed that thenanofiber-shaped sodium titanate phase is formed in such a fine scalebecause of the physically confined geometry of the host structure,TiO.sub.2 nanotubes. Since the nucleation and growth of the sodiumtitanate phase occurs on TiO.sub.2 which has the ring-shaped endmaterial facing outward with the tube wall thickness of onlyapproximately 15 nm, the sodium titanate phase growing from the hostsurface is likely to be on the order of or less than this dimension, asis actually observed. The process of forming a “Nano-inspiredNanostructure” can also be viewed as a hierarchical construction ofnanostructure, which can be important for nanostructural engineering,for example, for creation of catalyst structures with ultra-largesurface area.

The formation of bone-growth related material such as the calciumphosphate mineral, hydroxyapatite (Ca.sub.10(PO.sub.4).sub.6(OH).sub.2),is an important issue for orthopedic and dental implants. Bone is acalcium phosphate based mineral which contains approximately 70%hydroxyapatite-like material with the remainder consisting mostly ofcollagen.

The “Nano-inspired Nanostructure” formation as shown in FIG. 5 has beenfound to have a profound effect on hydroxyapatite formation. When theTiO.sub.2 nanotube surface covered with “Nano-inspired Nanostructure” ofsodium titanate is subjected to the SBF (simulated body fluid) solutionfor formation of hydroxyapatite, it is seen that it takes less than oneday soaking in SBF to have a complete coverage of the sample surfacewith hydroxyapatite. The formation of hydroxyapatite in the TiO.sub.2nanotube surface containing sodium titanate nanofibers is significantlyaccelerated as compared with the same TiO.sub.2 nanotube surface butwithout the sodium titanate nanofibers. In the latter case, it tookapproximately 7 days for formation of detectable amount ofhydroxyapatite, as compared with just one day for the sample coveredwith sodium titanate nanofibers.

As is evident from FIG. 5C, the hydroxyapatite formed is by itselfnanostructured with a nanofiber morphology resembling that of the sodiumtitanate. The nanofiber feature size of the hydroxyapatite phase formedis approximately 25 nm average diameter. It appears that the nanofiberhydroxyapatite nucleated and grew from the nanofiber sodium titanateprecursor. The approximately 25 nm average diameter of the nanofiberhydroxyapatite is somewhat coarser than its precursor sodium titanate(approximately 8 nm) as might be anticipated for the extended (1 day)exposure to SBF. The nanofiber hydroxyapatite of such a dimension is, tothe best of our knowledge, the smallest feature hydroxyapatite reportedso far.

Example 3 Osteoblast Cell Growth on Nanotubes of the Invention

In order to estimate the effect of having an extremely finenanostructure such as the vertically aligned TiO.sub.2 nanotubes on cellgrowth behavior, an osteoblast cell growth on TiO.sub.2 nanotubes wasperformed. The results demonstrate (indicate) that the introduction ofnanostructure significantly improves bioactivity of implant and enhancesosteoblast adhesion and growth. An adhesion of anchorage-dependent cellssuch as osteoblasts is a necessary prerequisite to subsequent cellfunctions such as synthesis of extracellular matrix proteins, andformation of mineral deposits. In general, many types of cells besidethe osteoblast cells remain healthy and grow fast if they arewell-adhered onto a substrate surface, particularly a nanostructuresurface of this invention, while the cells not adhering to the surfacetend to stop growing.

All the experimental specimens (0.5.times.0.5 cm.sup.2) used for celladhesion assays were sterilized by autoclaving. A pure Ti sheet polishedby emery paper (#600 grit size) and chemically cleaned was used as acontrol group sample. For cell adhesion studies, MC3T3-E1 osteoblastcells (rat cells of the type CRL-2593, sub-clone 4. ATCC, Rockville,Md.) were used. Each 1 mL of cells was mixed with 10 ml of alpha minimumessential medium (.alpha.-MEM) in the presence of 10% fetal bovine serum(FBS) and 1% penicillin-streptomycin. The cell suspension was plated ina cell culture dish and incubated under 37.degree. C., 5% CO.sub.2environment. When the concentration of the MC3T3-E1 osteoblastic cellsreached approximately 3.times.10.sup.5 cells/ml, they were seeded ontothe experimental substrate of interest (Ti O.sub.2 or Ti) which werethen placed on a 12-well polystyrene plate, and stored in a CO.sub.2incubator for 2, 12, 24 or 48 hrs to observe cell morphology and countviable attached cells as a function of incubation time. Theconcentration of the cells seeded onto the specimen substrate wasapproximately 5.times.10.sup.4 cells/ml.

After the selected incubation period, the samples were washed with 0.1 Mphosphate buffer solution (PBS) and distilled water, respectively, andfixed with 2.5% glutaraldehyde in 0.1 M PBS for 1 hr. After fixing, theywere rinsed three times with 0.1 M PBS for 10 min. For microscopicexamination of cell structures and morphologies, the samples weredehydrated in a graded series of alcohol (50%, 75%, 90% and 100%) for 10min and subsequently dried by supercritical point CO.sub.2. Thedehydrated samples were sputter-coated with approximately 2 nm thickgold for SEM examination. The morphology of TiO.sub.2 nanotubes as wellas that of the adhered cells was observed using SEM and TEM. In thequantitative assay, the adhered cells on sample surface were countedfrom back-scattered SEM images.

Shown in FIG. 6A-B are comparative SEM micrographs of the MC3T3-E1 cellscultured on pure Ti vs TiO.sub.2 nanotubes. After approximately 2 hoursof incubation, the osteoblast cells cultured on Ti surface, stillremained in their original round shape, whereas the cells cultured onTiO.sub.2 nanotubes attached onto the surface and started to spread byfilopodia. It is well known that pure Ti has a few nm thick, nativeTiO.sub.2 passivation layer which eventually causes the adhesion ofosteoblastic cells, albeit at a much slower speed than the nanotubesurface investigated in this work. It took approximately 12 hrs for anoticeable adhesion and propagation of the osteoblast cells to takeplace on Ti as shown in FIG. 6A. The growths of cells and propagation offilopodia are compared for the Ti sample (FIG. 6A) versus the TiO.sub.2nanotubes (FIG. 6B) after 12 hrs and 2 hrs of incubation, respectively.

As discussed earlier, micrometer-sized bioactive materials (such as ahydroxyapatite layer coated on Ti implant surface) tend to exhibitinterfacial failures, due to the much higher interfacial stress build-upbetween the dissimilar materials and also due too the lack of strongchemical bonding or the absence of sharing of common element speciesbetween the implant and the coating. The vertically aligned TiO.sub.2nanotube coating as described herein has the following structuraladvantages for reduced interfacial failure. [0081]i. The exemplaryvertically aligned TiO.sub.2 nanotube coating is fabricated to be thin,e.g., less than about 800 nm, or less than about 400 nm. [0082] ii. Thecoating has a strong chemical bonding on the Ti substrate as theTiO.sub.2 nanotube coating was prepared via chemical process, and sincea common element of Ti is shared by the substrate and the coating.[0083] iii. The TiO.sub.2 nanotube structure of FIGS. 1-3 is made to benot continuous but is discrete, with a gap between adjacent nanotubes ofapproximately 15 nm. The desired lateral gap dimension is in the rangeof about 2-100 nm, or about 5-30 nm. Such a lateral sub-division of ananotube array structure is important for minimizing the interfacialstresses between two dissimilar materials joined together, with the twomaterials involved often having substantially different crystalstructure, lattice parameter, and coefficient of thermal expansion.

It has experimentally been confirmed that the vertically alignedTiO.sub.2 nanotubes are strongly adherent to the Ti metal base, as itwas very difficult to remove the nanotubes from the Ti surface byattempting to delaminate or scrape off or by bending of the Tisubstrate. Such a strongly bonded and stable bone-promoting coating isimportant, especially in consideration of possible interference byfibroblast cells during bone growth near the Ti implants. It is wellknown that fibroblast cells are prone to attach on smooth surface layerin contrast to the osteoblastic cells which can attach well on roughsurface. (see e.g. Salata, Jour. of Nanobiotechnology (2): 3 (2004)).Once an opportunity and time is given for the fibrous tissues to form atthe boundary interface between the implant and the growing bone, thesetissues keep osteoblasts from adhering onto the surface of Ti implant,causing the undesirable loosening of the Ti implant. A rapid and strongadhesion of osteoblasts on implant surface is therefore an essentialfactor for successful bone growth.

In addition to the advantages in mechanical properties, the gaps presentbetween adjacent TiO.sub.2 nanotubes may also be useful as a pathway forcontinuous supply of the body fluid with ions, nutrients, proteins, etc.This is likely to contribute positively to the health of the growingcells. In the absence of such pathways, the proliferating cells willeventually completely cover the bioactive implant material surface, andthe bottom surface of the growing osteoblast cells would then have verylimited access to body fluid.

Presented in FIG. 7A-B are SEM micrographs showing the growth andadhesion of the osteoblast cells (after 2 hrs) on vertically nanoporousTiO.sub.2 nanotubes. The micrographs clearly indicate that the filopodiaof propagating osteoblast cells actually go into the vertical nanoporesof the TiO.sub.2 nanotubes. The observed rapid adherence and spread ofosteoblastic cells cultured on TiO.sub.2 nanotubes could be caused bythree reasons. First, vertically aligned TiO.sub.2 nanotubes exhibitenormously larger surface areas than the flat Ti surface. Second, thepronounced vertical topology contributes to the locked-in cellconfiguration. Thirdly, the pathway in-between TiO.sub.2 nanotube arrayscan allow the passage of body fluid and act as the supply/storage routeof nutrient, which is an essential biological element for cell growth.

FIG. 8A-C represents the comparative back-scattered SEM micrographs ofthe cells cultured on 8A pure Ti, 8B amorphous TiO.sub.2 nanotubes, and8C anatase TiO.sub.2 nanotubes after 48 hrs of incubation. It is evidentthat adhesion and growth of the MC3T3-E1 osteoblast cells issignificantly accelerated on TiO.sub.2 nanotubes, and more so on anataseTiO.sub.2 nanotubes, as compared to the amorphous TiO.sub.2 nanotubes.The plot of the number of adhered cells as a function of culture period,FIG. 9, clearly confirms this trend, with the speed of cell adhesion andgrowth on anatase TiO.sub.2 nanotubes being significantly higher after48 hr culture, by as much as approximately 400% as compared to the Tisurface. It is noted that at the early stage, (e.g., after 2 hrs.incubation) there was no significant statistical difference in the dataamong the three surfaces investigated. However, the number of attachedcells on the TiO.sub.2 nanotubes dramatically increases as the culturetime is extended to 12, 24 and 48 hrs.

The accelerated cell growth on vertical TiO.sub.2 nanotube array is notrestricted to the osteoblast cells. A similar behavior is seen withother cells, for example, it has been found that stem cell growth issubstantially accelerated on identical TiO.sub.2 nanotube array. This isshown in FIG. 10A-B as comparative back scattered electron SEM images.The cells used were bone marrow-derived, adult rat mesenchymal stemcells grown on plain flat Ti for 12 hr incubation, FIG. 10A, and onanatase TiO.sub.2 nanotubes for identical incubation period. As isevident from FIG. 10, the stem cells adhere and grow much faster on thevertically aligned and laterally separated TiO.sub.2 nanotubes than onthe plain flat Ti.

In stem cell research and applications for curing of diseases, theenrichment of stem cells is a very critical issue. The stem cells areoften rare and the available quantity is not always sufficient for manyresearch and therapeutic uses. The discovery that the TiO.sub.2nanotubes can significantly accelerate the kinetics of stem cellproliferation is therefore important from a practical point of view.This exemplary vertically aligned TiO.sub.2 nanotube array can also beutilized for such accelerated proliferation of many other types ofcells.

The vertically aligned and laterally separated TiO.sub.2 nanotube arraysas described herein have many useful applications some of which arelisted below.

1. Orthopedic and Dental Implants with Accelerated Bone Formation

The invention provides orthopaedic (orthopedic) and dental implantscapable of accelerated bone formation. In one aspect, fast recovery ofTi implant patients is an important benefit of using a TiO.sub.2nanotube array of the invention. Ti or Ti alloy implants (for example,hip implants, knee implants, elbow implants, Ti rods for broken legs orarms, etc., of the invention) with the surface modified to have a layerof the exemplary adherent TiO.sub.2 nanotube array can be placed inhuman or animal body as illustrated in FIG. 11A-11C.

2. Multi-Functional Implants

The invention provides multi-functional implants comprising verticalpores of TiO.sub.2 nanotubes. The vertical pores of the TiO.sub.2nanotubes of the invention can be utilized as a reservoir of variousbiologically active agents such as therapeutic drugs, growth factors,proteins, enzymes, hormones, DNA, genes, antibiotics, antibodies,magnetic nanoparticles, and so forth. The nanosize pores of TiO.sub.2nanotubes of the invention, as compared to microsized pores, have anadvantage of being able to keep the stored biological agents much longerand allow slower release over a longer period of time. Multifunctionalorthopedic or dental Ti implants of the invention can also continuouslysupply biological agents like a growth factor or bone morphogenicprotein (BMP) that can be slowly released from the TiO.sub.2 nanotubesurface layer can be much more efficient than a simple implant material.In one aspect, slow release of antibiotics (such as penicillin,streptomycin, vancomycin) can prevent infections near the implant. Sinceboth Ti and TiO.sub.2 are biocompatible, the implant (not necessarilybone-related implants, but including other implants of the invention)can be pre-filled with one or more types of drugs can be used as asource of slow drug release within a human body, for example fortreatment of cancer.

3. Externally Controllable Drug Release Devices of the cells isrelatively small. Each of the detection elements in FIG. 17, whichcontains a multiplicity of TiO.sub.2 nanotubes on which various types ofcells to be analyzed, are placed and allowed to rapidly proliferate to asufficient number for easy detection.

For analysis of cell types, various exemplary techniques, FIG. 18A-18C,can be used including 18A optical detection of morphology and size(using a microscope, fluorescent microscope, or CCD camera sensing offluorescent or quantum dot tagged cells), 18B chemical or biologicaldetection (e.g., based on signature reactions), 18C magnetic sensordetection (e.g., by using magnetically targeted antibody and itsconjugation with certain types of antigens).

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention. Forexample, the materials involved do not have to be Ti oxide nanotubes onTi-based metals, as the nanotubes and the substrate that the nanotubesare adhered to can be other biocompatible materials or non-biocompatiblematerials coated with biocompatible and bioactive surface layer such asTi, or coated with biocompatible but bio-inert surface layer such asnovel metal or polymer layer.

II. Artificially Engineered Biocompatible Nanotube and Nanopore Arrayfor Cell and Bone Growth and Method for Fabricating Thereof

This aspect of the invention provides template devices comprisingartificially engineered nanotube and nanopore structure covered withbiocompatible material such as TiO.sub.2. Also provided are artificialtissues and organs comprising theses template devices with sufficientand appropriate cells to provide a functional organ or tissue, e.g.,including connective tissue cells, liver cells, bone cells, kidneycells, blood vessel cells, skin cells, periodontal cells, odontoblasts,dentinoblasts, cementoblasts, enameloblasts, odontogenic ectomesenchymaltissue, osteoblasts, osteoclasts, fibroblasts, and other cells andtissues involved in odontogenesis or bone formation, and/or stem cells.

Also provided are methods for enhanced culturing of two-dimensionallyand three-dimensionally configured cells/organs using these templatedevices of the invention. These structures of the invention inducestrong cell adhesion and significantly enhance the formation kinetics ofcells.

The three-dimensionally placed nano-gaps between aligned nanotubes andnanopores allow a continuous supply of various nutrients to the growingcells. The nanotube and nanopore structure can be either retractablefrom or permanently kept in the grown cells. This 3-dimensional cell andtissue culture device of the invention, which can comprise storedbiological agents within the nanotube reservoirs, can improve growth ofconfigured and healthy cells including liver cells, bone cells, kidneycells, blood vessel cells, skin cells, periodontal cells, odontoblasts,dentinoblasts, cementoblasts, enameloblasts, odontogenic ectomesenchymaltissue, osteoblasts, osteoclasts, fibroblasts, and other cells andtissues involved in odontogenesis or bone formation and/or stem cells,to name just a few examples. The 3-dimensional cell and tissue culturedevice of the invention, and cultured cells derived from these devices,can be useful for rapid supply of needed cells for research anddevelopment, e.g., for drug development or testing or for therapeutics,or for preparation of partial or full implant organs, for externallycontrollable drug release and therapeutic treatments, for efficienttoxicity testing of drugs and chemicals, and/or for diagnosis/detectionof disease or forensic cells.

SUMMARY

The invention provides arrays comprising a solid substrate and aplurality of vertically aligned, laterally spaced, nanotubes associatedwith the substrate, wherein each nanotube comprises a nanopore. In oneaspect, the outer diameter of each nanotube is between about 10 to 1000nm, or, the outer diameter of each nanotube is between about 30 to 300nm, or the outer diameter of each nanotube is between about 60 to 200nm.

In one aspect, a nanopore of each nanotube comprises a diameter of (atleast) about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75% or more, of the outer diameter of the nanotube.

In one aspect, the height of at least one, several or all (each)nanotube is about 40 to 1000 nm, or, about 100 to 400 mm, or 200 to 800nm, and the like.

In one aspect, the aspect ratio of each nanotube is less than about 15,less than about 14, less than about 13, less than about 12, less thanabout 11, less than about 10, or less than about 9, less than about 8,less than about 7, less than about 6, or less than about 5.

In one aspect, the alignment angle of the vertically aligned nanotubesis between about 0 to 50, 45, 40, 35, 30, 25, 20, 15 or 10 degrees offthe vertical direction, or, the alignment angle of the verticallyaligned nanotubes is about 0 to 60 degrees off the vertical direction.

In one aspect, adjacent, vertically aligned nanotubes are laterallyspaced from between about 2 to 100 nm, or between about 5 to 30 nm, orbetween about 10 to 90 nm and the like.

In one aspect, adjacent, the arrays comprise a biocompatible surface.The biocompatible surface can comprise a compound selected from thegroup consisting of Ti. Ti oxide, ceramic, noble metals, and polymermaterials.

The arrays of the invention can further comprise two-dimensionallyand/or three-dimensionally cultured cells selected from the groupconsisting bone cells, liver cells, kidney cells, blood vessel cells,skin cells, periodontal cells, odontoblasts, dentinoblasts,cementoblasts, enameloblasts, odontogenic ectomesenchymal tissue,osteoblasts, osteoclasts, fibroblasts, and other cells and tissuesinvolved in odontogenesis or bone formation and/or stem cells, to namejust a few examples, and other human or animal organ cells. The arraysof the invention can further comprise two-dimensionally and/orthree-dimensionally cultured organs, wherein the organ comprises one ormore cells selected from the group consisting of bone cells, livercells, kidney cells, blood vessel cells, skin cells, periodontal cells,stem cells, and other human or animal organ cells.

The arrays of the invention can further comprise a means for retractingor withdrawing or removing (and the like) the nanotubes from thethree-dimensionally cultured cells such that only the cells and/ororgans are left. In one aspect, the three-dimensionally cultured cellsor organs are permanently or semi-permanently associated with thenanotubes of the array.

The invention provides methods for accelerating the growth of cells, themethod comprising contacting the cells with an array of the invention inthe presence of a nutrient fluid suitable for sustaining growth of thecells. In one aspect of the method, cell growth is accelerated by about25%, 50%, 75%, 100%, 150%, 200%, 250% or 300% or more. In one aspect,the nutrient fluid is supplied under the growing cells through thespacing between the parallel nanotubes.

The invention provides arrays comprising a solid substrate and aplurality of vertically aligned, laterally spaced, nanotubes associatedwith the substrate, wherein each nanotube comprises at least onenanopore and the array comprises “dental” cells (e.g., odontoblasts,dentinoblasts, cementoblasts, enameloblasts, odontogenic ectomesenchymaltissue, osteoblasts, fibroblasts, and other cells and tissues involvedin odontogenesis) suitable for odontogenesis, and implantation andregeneration of dental tissue, e.g., teeth, dentin, cementum, enamel orsupporting structures, in a subject, e.g., a human.

The invention provides arrays comprising a solid substrate and aplurality of vertically aligned, laterally spaced, nanotubes associatedwith the substrate, wherein each nanotube comprises at least onenanopore and wherein the array comprises orthopaedic cells suitable forimplantation and regeneration of bone and/or joint tissue in a subject.

The invention provides arrays comprising a solid substrate and aplurality of vertically aligned, laterally spaced, nanotubes associatedwith the substrate, wherein each nanotube comprises at least onenanopore and wherein the array comprises one or more biologically activeagents selected from the group consisting of therapeutic drugs, growthfactors, proteins, collagens, stem cells, enzymes, hormones, DNA's,genes, antibiotics, antibodies, and magnetic nanoparticles.

The invention provides arrays comprising a solid substrate and aplurality of vertically aligned, laterally spaced, nanotubes associatedwith the substrate, wherein each nanotube comprises at least onenanopore and wherein the array comprises a colloidal compositioncomprising magnetic nanoparticles interspersed with a biological agentselected from the group consisting of therapeutic drugs, cancer drugs,growth factors, proteins, collagens, stem cells, enzymes, hormones,DNA's, genes, antibiotics, and antibodies.

The invention provides methods for selectively releasing a biologicalagent in a subject, the method comprising implanting an array of theinvention in a subject and contacting the array with ultrasonic ormagnetic agitation of the colloidal composition, wherein the biologicalagent is released from the array. In one aspect, the magneticnanoparticle is selected from the group consisting of iron-oxideparticles of magnetite (Fe.sub.3O.sub.4) or maghemite(.gamma.-Fe.sub.2O.sub.3). In one aspect, the nanoparticles are about 5to 50 nm in average diameter, or about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 75, 100 or more nm in average diameter.

In one aspect, the magnetic agitation comprises external stimulation ofthe magnetic nanoparticles by alternating current (AC) magnetic field.The biological agent can be released from the array by mechanicalagitation/movement of the magnetic particles or by heating of thecomposition resulting from the AC magnetic field.

In one aspect, the array or chip of the invention further comprisesnanotubes configured with narrow passage caps at the upper end of thenanotube, wherein the narrow passage inhibits the release of thebiological agent from the nanopore. In one aspect, the “cap”, ornarrowed passage, is deposited by oblique incident sputter deposition ona stationary or a rotating substrate.

The invention provides methods for treating a cell proliferationdisorder, the method comprising: a) implanting an array of the inventioninto a subject (e.g., a human in need thereof), wherein the array isimplanted at or near the site of a cell proliferation disorder; and b)contacting the array with magnetic agitation, wherein the agitationaccelerates biological agent release and provides magnetic hyperthermiatreatment at the site of implantation.

The invention provides systems for growing and harvesting selectedcells, the system comprising: a) an array of the invention operablyassociated with a device for removing the cells or tissue from thearray; and b) a computer operably associated with a), wherein thecomputer comprises instructions for automatically contacting the cellswith a suitable growth media and for harvesting the mature cells. In oneaspect, the cells comprise connective tissue cells (e.g., fibroblasts),bone cells, liver cells, pancreatic cells (e.g., beta cells), kidneycells, blood vessel cells, odontoblasts, dentinoblasts, cementoblasts,enameloblasts, odontogenic ectomesenchymal tissue, osteoblasts,osteoclasts and/or skin cells. In one aspect, the cells are embryonic oradult stem cells. In one aspect, the cells are removed bytrypsinization. In one aspect, the cells are harvested using mechanicalmeans such as suction and centrifugal process, or optionally incombination with trypsinization.

The invention provides methods for the diagnosis, prognosis or detectionof a disease or condition (e.g., allergies, toxicities, geneticcondition) comprising implanting an array or chip, e.g., a biochip,comprising an array of the invention in a subject in need thereof (e.g.,a human), or, using the array or chip in situ or in vitro. In oneaspect, the array comprises a cell or tissue relevant for the diagnosis,prognosis or detection.

The invention provides a method for detecting a biological agent, e.g.,a toxin, a poison or a biological warfare agent, e.g., anthrax, themethod comprising use of an array or chip, e.g., a biochip of theinvention, e.g., providing an array comprising an X-Y matrix subdividedarray of nanotubes (e.g., comprising a plurality of nanotubes), eachnanotube (or set of nanotubes, e.g., each row, or a cluster of tubes)comprising a specific type of cell for analysis.

The invention provides a biomimetic array, or artificially constructedcell culture, comprising liver cells for performing drug/chemicaltoxicity testing, the array comprising an array of the inventioncomprising liver cells (e.g., parenchymal cells, or hepatocytes,endothelial cells, adipocytes, fibroblastic cells and/or Kupffer cells)for evaluation of new drugs for testing of safety and toxicity issues.The invention provides methods for evaluating the toxicity of acompound, e.g., a drug, small molecule, food additive or food coloring,cosmetic, pesticide, natural product, biological warfare agent, and thelike, using a biomimetic array of the invention. In one aspect, thearray is contacted with a test material such as a chemical (e.g., adrug, small molecule, food additive or food coloring, cosmetic,pesticide), a polymer, an injection fluid, a biological agent (e.g., atoxin, biological warfare agent, e.g., a gas, such as Mustard gas,Sarin).

The invention provides methods for manufacturing an array of theinvention, e.g., using the exemplary methods described herein.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

DETAILED DESCRIPTION

The invention provides artificially engineered biocompatible nanotube-and/or nanopore-comprising arrays comprising alternative materials oralternative fabrication techniques for biocompatible nanotube andnanopore array for rapid growth of cells/tissues/bones.

The invention provides enhanced culturing of two-dimensionally andthree-dimensionally configured cells/organs/bones using template devicescomprising artificially engineered nanotube and nanopore structurecovered with biocompatible material such as TiO.sub.2. This exemplarystructure induces a strong cell adhesion and significantly enhances theformation kinetics of cells. In these exemplary designed, fabricated andaligned nanotubes of the invention, the practical limit ofanodization-based processing (which produce a rather limited TiO.sub.2nanotube diameter, about 100 nm regime) can be overcome. In theseexemplary designed nanotubes of the invention, a desired diameter can begreater than about 100 nm, and in one aspect the cell growth can beaccelerated and optimized. In one aspect, the three-dimensionally placednano-gaps between the nanotubes can allow a continuous supply of variousnutrients to the growing cells. The nanotube and nanopore structure ofthe invention can be either retractable from the cultured cells or canbe permanently kept in the grown cells. One exemplary cell and tissueculture device of the invention comprises stored biological agentswithin the nanotube reservoirs, can improve growth of configured andhealthy cells, including for example, liver cells, bone cells, kidneycells, nerve cells, blood vessel cells, skin cells, periodontal cells,stem cells. Such cultured cells can be useful for rapid supply of neededcells for R&D or therapeutics, for preparation of partial or fullimplant organs, for externally controllable drug release and therapeutictreatments, for efficient toxicity testing of drugs and chemicals, andfor diagnosis/detection of disease or forensic cells.

In one aspect, nanotubes of the invention are fabricated in such a waythat they have a controlled, pre-determined diameter, and/or aninter-nanotube gap spacing, and/or a predetermined height (as comparedwith the typical anodized TiO.sub.2). In one aspect, the surface of thenanotube array, or in one aspect—at least the top surface, or all thesurfaces, are coated with a biocompatible layer, such as Ti orTiO.sub.2, or other biocompatible metallic, ceramic, polymer, orbiomolecule coating.

In one aspect, the nanotube alignment comprises a vertical alignmentwith a open top pore, which in some applications may be crucial for bioimplant and related applications, as disclosed herein. In one aspect,the open top of the nanotubes allows the penetration of the cells intothe nanopore cavity for good adhesion. In one aspect, the open top ofthe nanotubes also allows easy supply of the biological agents stored inthe nanopores. In one aspect, the cells that adhere well to a surfacegenerally stay healthy and grow fast, while the cells that do not adhereexhibit reduced or minimal growth. In one aspect, the desired angle ofvertical alignment is within than about 10, 20 or 30 degrees off theperpendicular axis.

In one aspect, the nanoscale Ti oxide structures of the inventioncomprise Ti and Ti alloys that are corrosion resistant, light, yetsufficiently strong for load-bearing, and are machinable. They are oneof the few biocompatible metals which osseo-integrate (direct chemicalor physical bonding with adjacent bone surface without forming a fibroustissue interface layer); thus, the nanoscale Ti oxide structures of theinvention can directly bond, by e.g., chemical and/or physical bonding,with adjacent bone surface without forming a fibrous tissue interfacelayer. Thus, the nanoscale Ti oxide structures of the invention can beused as orthopaedic (orthopedic) and dental implants, e.g., with thedevices described in the Handbook of Biomaterial Properties, edited byJ. Black and G. Hasting, London; Chapman & Hall, 1998; or, Ratner etal., Biomaterials Science, San Diego, Calif.: Academic press; 1996.

While the invention is not limited by any particular mechanism ofaction, the bioactivity of Ti, such as the relatively easy formation ofhydroxyapatite type bone mineral on Ti, is primarily caused by theoccurrence of Ti oxide on the surface of Ti and its alloys. Variouscrystal structures of Ti oxide can be used in making the compositions ofthe invention, e.g., the anatase phase, which is known to be better thanthe rutile and other phases (however, the rutile and other phases canalso be used), as described, e.g., by Uchida et al. Journal ofBiomedical Materials Research (2003) 64:164-170.

Surface treatments such as roughening by sand blasting, formation ofanatase phase TiO.sub.2, hydroxyapatite coating, or other chemicaltreatment can also be utilized to further improve the bioactivity of Tisurfaces of the invention, and, in various aspects, to enhance cell,tissue and/or bone growth.

TiO.sub.2 phase can be prepared by various techniques such as sol-gelmethod, electrophoretic deposition and anodization. See, e.g., Lakshmi,et al., Chemistry of Materials, Vol. 9, page 2544-2550 (1997), Miao, etal., Nano Letters, Vol. 2, No. 7, page 717-720 (2002); Gong, et al.,Journal of Materials Research, Vol 16, No 12, page 3331-3334 (2001). Inone aspect, a TiO.sub.2 phase can be prepared by various techniques suchas sol-gel method (see, e.g., U.S. Pat. No. 7,014,961);photolithographic patterning of a photoresist layer by pattern-wiseexposure to short-wavelength ultraviolet light through a pattern-bearingphotomass, as described in U.S. Pat. No. 6,593,034; electrophoreticdeposition and anodization. See, e.g., Lakshmi, et al. (1997) Chemistryof Materials, 9:2544-2550; Miao, et al. (2002) Nano Letters 2:717-720;Gong, et al. (2001) J. Materials Res. 16:3331-3334; Macak (2005) Chem.Int. Ed., 44:7463-7465.

In one aspect, fabrication of vertically aligned TiO.sub.2 nanotubes onTi substrate can be done by anodization process, and the inventionincludes use of titanium oxide nanotubes for cell, tissue and/or bonegrowth or other bio application. Patients who go through Ti implantoperations for repair of hip joints, broken bones, or dental implantsoften have to wait for many months of slow bone growth recovery beforethey are cured enough to get off the confinement on a bed or crutchesand have a normal life. Accelerated bone growth would thus be verybeneficial for such patients. Therefore, the biocompatiblenanostructures in desirable configuration for enhanced bone growth andother cell growth is useful for a variety of bio applications.Furthermore, biocompatible nanostructures of the invention can be madeto serve multifunctional roles to additionally accelerate tissue and/orcell growth (e.g., bone). In one aspect, they are used in orthopaedicand/or dental applications.

In one aspect, coating of bioactive materials such as hydroxyapatite andcalcium phosphate on Ti surface is used to make the Ti surface morebioactive. See, e.g., Shirkhanzadeh et al, Journal of Materials ScienceLetters, Vol. 10, page (1991), de Groot et al., Journal of BiomedicalMaterials Research, Vol. 21, page 1375-1381 (1987), and Cotell et al.,Journal of Applied Biomaterials, Vol. 8, page 87-92 (1992). The coatingtechniques of the invention, and the resultant structures of theinvention, in contrast to flat and continuous coatings, fail less byfracture or delamination at the interface between the implant and thecoating as an adhesion failure, or at the interface between the coatingand the bone, or at both boundary interfaces. The coating techniques ofthe invention, and the resultant structures of the invention, incontrast to thick film coatings, introduce fewer interface stresses atthe substrate-coating interface; see, e.g., Yang, et al., Journal ofBiomedical Materials Research, Vol. 36, page 39-48 (1997). In oneaspect, the interface is bonded with an improved and integratedstructure, for example, with a locked-in configuration having a muchincreased adhesion area, and as a discrete, less continuous layer tominimize interface stress and de-lamination. In one aspect, acceleratedhydroxyapatite and bone growth is accomplished on a mechanicallyadherent TiO.sub.2 nanotube surface of the invention on a Ti substrate;see, e.g., Oh (2005) Biomaterials 26:4938-4943.

The invention provides compositions and methods for the fast growth andsupply of cells and tissues, such as liver cells, kidney cells, bloodvessel cells, and stem cells; and this can be crucial for many potentialtherapeutic and diagnostic applications. Liver cells can be cultured forpartial or fall liver implantation or for hepatic support of patientsprior to liver transplant operation, as described, e.g., in US PatentApplication Pub. No. US 2002/0072116 A1 by Bhatia et al.

The invention provides compositions and methods for the fast detectionand diagnosis of diseased or abnormal cells, or cells exposed to ormodified by a biological warfare agent, for example, compositions andmethods of the invention can be used in fast detection and diagnosisbiological or chemical warfare agents (e.g., gases, such as Sarin,mustard gas, etc., toxins), epidemic diseases, anthrax, influenza (e.g.,the so-called “bird flu), or SARS.

In one aspect, the compositions and methods use a very small or tracequantity of available cells; this can be accomplished if the cell growthspeed can be accelerated. In one aspect, an accelerated bone and cellgrowth using vertically aligned and laterally separated TiO.sub.2nanotube arrays are used, with the emphasis on two-dimensional cellgrowth.

Because desired cell growths for implant and other applications mayrequire three-dimensionally configured cells, the invention providesmethods and devices for producing such three-dimensionally culturedcells. In one aspect, the invention provides methods and devices forfast growth and supply of functional cells, such as liver or kidneycells, without a deterioration of their function(s), which can becritical for many organ (e.g., kidney or liver) therapeuticapplications. In one aspect, the invention provides a bio-artificialliver or bio-artificial kidney support device. In one aspect, nanotubulestructures of the invention are useful as substrates for efficient organcell (e.g., kidney or liver cell) culture devices if the cells (e.g.,liver cells) can be grown quickly in vitro without a major loss of theircapability for complex functions.

In one aspect, the invention provides structures that are self-assembledin a simple anodization process, which in one aspect provides a viable,three dimensionally configured surface for enhanced cell growth and/orculture in vitro, in situ, ex vivo and in vivo. In controlling thespecifics of nanotube diameter, spacing and height (the inventionprovides compositions having alternative nanotube diameters, spacing andheights), the compositions of the invention also can be used in variousprocessing steps, different organ systems, varying combination of cells,and the like, as described herein.

This invention provides 2-dimensional and 3-dimensional cell culturedevices comprising artificially patterned and shaped nanotube array(s)with a biocompatible coating, such as Ti or TiO.sub.2. These structuresof the invention enable accelerated growth of functional organ cellssuch as liver or kidney cells as well as other structural cells such asblood vessel cells, enzyme secretion vessels, periodontal, bone, teeth,or other hard tissue cells growth. The availability of such culturedcells can be useful for a variety of applications including i)organ-related therapeutic medical treatment including liver or kidneydisease treatment, ii) orthopaedic, dental or periodontal processes,iii) supply of cells for various research or therapeutic purposes, andiv) disease diagnostic or toxicity testing of new drugs or chemicals.

Referring to the drawings, FIG. 1A schematically illustrates exemplarydevices comprising exemplary artificially configured nanotube array. Thenanotubes are fabricated in such a way that they have controlled,pre-determined diameter, inter-nanotube gap spacing, and height. Thesurface of the nanotube array, at least the top surface, or in oneaspect on several or all the surfaces, are coated with a biocompatiblelayer such as Ti or TiO.sub.2, or other metallic, ceramic, polymer, orbiomolecule coating.

The nanotubes for the invention's devices can have any desireddimension, e.g., between about 10 to 1000 nm in diameter, or betweenabout 30 to 300 nm, or anywhere in the nanoscale dimension of betweenabout 60 to 200 nm in diameter. The desired heights of the tubules canbe determined in part by the desired aspect ratio, as relatively shortheight with an aspect ratio of less than 10, or less than 5, in thisaspect is preferred for ease of storing and eventual dispensing of drugsor biological agents intentionally placed within the tubule cavity. Thisnanotubule arrangement and size can maintain the function of the cellsbeing cultured, e.g., kidney, liver or other cells, thus help theproliferation of these cells.

Exemplary heights of nanotubes can be between about 40 to 1000 nm, orbetween about 100 to 400 nm. In one aspect, a vertical alignment withopen top pore is crucial for bio-implant or related applications, e.g.,as in one aspect an open top a the nanowire allows the penetration ofthe cells into a nanopore cavity for good adhesion, as illustrated inthe exemplary FIG. 1B; this also allows easy supply of the biologicalagents stored in the nanopores.

Because it is well known that the cells that adhere well to a surfacegenerally stay healthy and grow fast, while the cells that do not adhereexhibit reduced or minimal growth, in one aspect the desired angle ofvertical alignment is within about 10, 20, 30 or 40 or more degrees offthe perpendicular axis.

Exemplary micro-structural features and advantages of nanotube arraystructure are described in FIGS. 2 to 4, for the exemplary anodized,self-assembled TiO.sub.2 structures of the invention. These scanningelectron microscopy (SEM) and transmission electron microscopy (TEM)images show exemplary micro-structural features comprising verticallyaligned, biocompatible TiO.sub.2 with a typical dimension of the hollownanotubes in FIGS. 2 and 3 being approximately 100 nm outer diameter andapproximately 70 nm inner diameter with approximately 15 nm in wallthickness, and approximately 250 nm in height.

Exemplary nanotube array structures of the invention provide for healthycell growth by providing a continuous supply of nutrients, includingproteins, mineral ions, fluid, etc. to the cell through the flow of bodyfluid. The gap (spacing) between adjacent exemplary nanotubules in FIGS.1-3 serves such a function of allowing the body fluid to continuouslypass through and supply nutrients to the bottom side of the growingcells. The desired gap between the nanotubules in the exemplarystructure of FIG. 1 is in the range of between about 2 to 100 nm, orbetween about 5-30 nm. In some aspects, too small a gap reduces theeffectiveness of nutrient body fluid flow while too large a gap can posea danger of reduced mechanical stability in the event of vertical orlateral stress or pressure.

Accelerated Cell Growth on TiO.sub.2 Nanotube Array

The invention provides nanotube structures that substantially improvecell adhesion and growth kinetics, an example of such an adhesiondepicted in the exemplary structure of FIG. 7. An adhesion ofanchorage-dependent cells such as osteoblasts is a crucial prerequisiteto subsequent cell functions such as synthesis of extracellular matrixproteins, and formation of mineral deposits. In general, many types ofcells beside the osteoblast cells remain healthy and grow fast if theyare well-adhered onto a substrate surface, while the cells not adheringto the surface tends to stop growing.

In one aspect, nanotube structures (e.g., on orthopaedic or dentalprostheses) can also comprise use of bone growth enhancing coatingscomprising micrometer-sized bioactive materials, such as ahydroxyapatite layer coated on Ti implant surface; noting that themicrometer-sized materials alone may exhibit interfacial failures due tothe much higher interfacial stress build-up between the dissimilarmaterials. Micrometer-sized materials alone also may fail due too thelack of strong chemical bonding or the absence of sharing of commonelement species between the implant and the coating. However, thesedisadvantages are mitigated by incorporation of the nanotube structuresof the invention.

Exemplary vertically or parallel-aligned nanotube arrays can have thefollowing structural advantages for reduced interfacial failure: (i)vertically or parallel-aligned nanotube arrays fabricated to be thin,e.g., less than 1000 nm, or less than 400 nm (ii) nanotube arrays madeas the same material as the base substrate material to ensure for strongbonding and mechanical stability of the nanotube array; and/or (iii)nanotube structures of FIG. 1 made to be not continuous but as discretestructures, e.g., with a gap between adjacent nanotubes of approximately15 nm.

In one aspect, lateral gap dimensions are in the range of between about2 to 100 nm, or between about 5 to 30 nm. In one aspect, this exemplarylateral sub-division of a nanotube array structures is important forminimizing the interfacial stresses between two dissimilar materialsjoined together, with the two materials involved often havingsubstantially different crystal structure, lattice parameter, andcoefficient of thermal expansion.

In one aspect, in addition to the advantages in mechanical properties,the gaps present between adjacent nanotubes may also be useful as apathway for continuous supply of the body fluid with ions, nutrients,proteins, etc. This may contribute positively to the health of thegrowing cells. In one aspect, in the absence of such pathways, theproliferating cells may completely cover the bioactive implant materialsurface and the bottom surface of the growing osteoblast cells wouldthen have very limited access to body fluid.

Presented in FIG. 7 are SEM micrographs showing the growth and adhesionof the osteoblast cells (after 2 hrs) on exemplary vertically orparallel-configured nanotube arrays, as an example of TiO.sub.2nanotubes. The micrographs clearly indicate that the filopodia ofpropagating osteoblast cells actually go into the vertical nanopores ofthe exemplary TiO.sub.2 nanotubes.

Noting the invention is not limited by any particular mechanism ofaction, the observed rapid adherence and spread of osteoblastic cellscultured on TiO.sub.2 nanotubes could be caused by three reasons. First,vertically aligned nanotubes exhibit enormously larger surface areasthan the flat Ti surface. Second, the pronounced vertical topologycontributes to the locked-in cell configuration. Thirdly, the pathwayin-between nanotube arrays can allow the passage of body fluid and actas the supply/storage route of nutrient, which is an essentialbiological element for cell growth.

Schematically illustrated in FIG. 19 are exemplary processing steps forguided anodization of Ti using pre-fabricated nano spot array forcontrolled TiO.sub.2 nanotube geometry. Various lithography techniquessuch as optical lithography (including laser lithography), electron beamlithography, ion beam lithography, self-assemble nanoparticlelithography, nano-imprint lithography, can be utilized to introduce anarray of small etched cavity or indented cavity on the surface of Ti orTi alloys. In one aspect, these pre-patterned spot locations serve as anucleation spots during the subsequent anodization process. Such aguided anodization process creates more uniformly spaced, pre-controlleddiameter and location of TiO.sub.2 nanotube array.

In one aspect, a patterned TiO.sub.2 nanotubule array is fabricated byan anodization process, for example, using the electrochemicalprocessing in a 0.5% HF solution at 20 V for 30 min at room temperature(RT). An inert electrode such as a platinum electrode can be used as thecathode. To crystallize the as-deposited, amorphous-structured TiO.sub.2nanotubes into the desired anatase phase, the as-made nanotubes can beheat-treated at approximately 400.degree. C. to 600.degree. C. foranywhere between about 0.1 to 10 hrs. In one aspect, the amorphousTiO.sub.2 nanotubes is crystallized to anatase phase by heat treatment,because an amorphous TiO.sub.2 phase tends to be more susceptible tobreakage by external stresses as compared to a crystalline phase. Theuse of the amorphous TiO.sub.2 phase nanotube array for specialapplications is not excluded, however.

Shown in FIG. 20 is an exemplary lithographical fabrication of gappednanotube array followed by coating with a biocompatible layer such as Tior TiO.sub.2, or various noble metal, ceramic, polymer or biomoleculelayer. Well known lithography materials and processes, such as the useof photoresist materials like PMMA, lithography beam exposure, selectiveetching (chemical or reactive-ion-etching), washing and drying can beutilized for fabrication of such a gapped nanotube array structure. Fordeposition of the biocompatible layer, physical vapor depositiontechniques such as evaporation, sputtering, laser ablation, chemicalvapor deposition, or chemical processes can be used. A slightly obliqueincident sputter or evaporation deposition can optionally be utilized tomake the coating of the nanotube inside wall easier. If desired, theside and the back surfaces of the nanotube-containing substrate can alsobe coated with the similar biocompatible layer.

FIG. 7 describes an alternative exemplary method of the invention forcreating biocompatible nanotube array comprising steps of lithographicalfabrication of nanocavity, inside wall deposition of nanotube materialsuch as Au, Pd, Pt, carbon, Ti or TiO.sub.2, for example, by obliqueincident sputtering. The matrix material can be then selectively etchedaway to leave only the nanotube array. For Al or Al-oxide matrix, NaOHsolution can be used for etching. For Si matrix, known Si etchingsolution including KOH can be used. In one aspect, the nanotube array sofabricated is then coated with a biocompatible layer such as Ti orTiO.sub.2, or various noble metal, ceramic, polymer or biomoleculelayer, for example, using DC or RF sputtering, or chemical processes. Ifthe nanotube array material itself is Ti or TiO.sub.2, the last coatingstep can of course be omitted.

FIG. 22 schematically illustrates an exemplary process of preparing achanneled array of parallel-aligned nano-cavities comprising a surfacecoating of biocompatible layers such as Ti or TiO.sub.2. Here thestructure is less complicated than the nanotube array structure in thatonly patterned and diameter-controlled nano-cavity array is present. Inone aspect, a channel structure is provided between the nano-cavities sothat the biological fluid and nutrients are continuously supplied to thegrowing cells for healthy and accelerated cell culture. This structurealso can be coated with a biocompatible layer such as Ti or TiO.sub.2,or various noble metal, ceramic, polymer or biomolecule layer, forexample, using DC or RF sputtering or chemical processes.

For these embodiments, the matrix material can be selected from Ti, Zr,Hf, Nb, Ta, Mo, W, and their oxides, or alloys of these metals andoxides. Other materials such as Si. Si oxide, Al, Al oxide, carbon,diamond, noble metals (such as Au, Ag, Pt and their alloys), polymer orplastic materials, or composite metals, ceramics or polymers can also beutilized to produce and use similar desired artificially generatednanotube or nanopore patterns. In one aspect, the surface of thenanotubes or nanopores are coated with biocompatible materialscomprising Ti and/or Ti oxide, or Zr, Hf, Nb, Ta. Mo, W and/or theiralloys or oxides of these metals and/or alloys, with a thickness of atleast 1, 2, 3, 4 or 5 or more nm and in one aspect the coating coverageof at least about at least 20%6, 30%, 40%, 50%, 606, 70%, 80% or more ofthe nanotube or nanopore surfaces.

Three-Dimensional Cell Growth

The invention also provides compositions and methods for 3-dimensionalcell, tissue and organ growth and maintenance, e.g., for organ or hardtissue replacements. In one aspect, a three-dimensional (3-D) growth ofcells with a substantial volume, rather than a thin, two-dimensional(2-D) surface coverage of cells, is desirable. The invention provides a3-D cell culture technique using the nanotube arrays with abiocompatible surface in a 3-D configuration. Referring to the drawings,FIG. 23 schematically illustrates an exemplary novel approach of theinvention. In one aspect, the growth base is made of subdivided andthree-dimensionally positioned Ti ribbons, plates, wires, rods asillustrated in FIG. 23A, which is in this example is made to beretractable so that the final cultured 3-D cells or organs do notcontain the Ti metal any more as might be desired for some applications,although Ti and TiO.sub.2 are well known to be biocompatible.

In one aspect, to obtain relatively large surface area for cell growth,the desired titanium metal ribbon thickness in FIG. 23A is in the rangeof between about 10.mu.m to 50,000.mu.m, or between about 25 to2,500.mu.m. In one aspect, the desired volume fraction of the metal forthe given targeted cell volume at the end of the planned cell cultureperiod is at least 5%, 10%, 20% or 30% in volume. Each of the ribbonsurface can be made to contain an aligned nanotube array with a desireddimension of nanotubes, as described above, e.g., a desired diameter ofbetween about 10-1000 nm, between about 30-300 nm, or between about 60to 200 nm in diameter; and the desired height can be in the range ofbetween about 40 to 1000 nm, or between about 100 to 400 nm; and thedesired angle can be vertical with an allowance of 10, 20, 30, 40 ormore degree variation off the perpendicular axis; and the desired gapdimension between adjacent nanotubes can be in the range of betweenabout 2 to 100 nm, or 5 to 30 nm.

In one aspect, the shape of these nanotubed ribbons is straight so thatthe metal arrays can be pulled out after desired volume of the cells arecultured, as illustrated in the exemplary FIGS. 23B and 23C. Thecultured growth can continue after the metal wire or ribbon arraytemplate structure is pulled out, and any minor surface damage or thethin empty gap created by the vacated template can be repaired/filled asillustrated in FIG. 23D. This exemplary three-dimensionally culturedcell volume in an accelerated and nutrient-supplied manner can be usefulfor a variety of applications, including making a partially artificialor a fully artificial tissue or organ, e.g., liver, kidney, bone,periodontal tissue, blood vessel cells, skin cells, stem cells, andother human or animal organ cells etc.

The 3-D cultured cells can be in any orientation, i.e., horizontal,vertical or upside down depending on specific needs, especially in exvivo or in vivo culture environment(s), for example, in the case oforgan implants in human, animal, or xenotransplantation of human organswhich are temporarily cultured in animals prior to human implantation.

Growth of Tube-Shaped Cells/Organs

An alternative embodiment of the invention comprises culturing3-dimensional, tube-shaped cells, such as blood vessel cells or fluid,enzyme and/or hormone secretion tubes, intestine tubes, nerve “tubes”(e.g., Schwann cells), and the like, using nanotube-comprisingcompositions and methods of the invention. For example, in one aspect,using artificially engineered nanotube array surfaces of this invention,the cells are cultured into a tube configuration; and in one aspect, theafter the cells are cultured into a tube configuration the rods arepulled out, leaving a ready made tubular cell structure (e.g.,multi-cellular structure) or a tubular cell shape. Such a tube-shapedcells or cell structure can be used for repairing tissues or organs,e.g., repairing damaged blood vessels, nerves, fluid, enzyme and/orhormone secretion tubes, fluid, intestine tubes, to name just a fewexemplary structures that can be built de novo, or used for the repairor reconstruction of tissues or organs, using the compositions andmethods of the invention.

Co-Culturing

Another embodiment of the invention comprises a 3-dimensional cellgrowth process of culturing at least two types of cells together. Humanor animal organs often contain more than one type of cells. Because ithas been recognized that a co-culture during growth of cells, such asbone, kidney or liver cells, is beneficial in obtaining a higher qualitycells (see, e.g., Begue (1983) Experimental Cell Res. 143:47-54; Angius(1988) J. Biochem. 252:23-28); US Patent application pub. No. US2001/0023073, Bhatia et al.), or a better functional organization ofcells-leading to a functional tissue or organ system, the inventionprovides compositions and methods comprising use of at least two typesof cells together. The invention also provides methods for co-culture ofdifferent cells using the compositions of the invention. e.g., formaking artificial organs or tissues, or for repairing or reconstructingorgans or tissues in vivo, ex vivo or in situ.

For example, in one aspect cells are co-cultured using artificiallyengineered nanotube array surfaces of the invention, and in one aspectat least two different types of cells are co-cultured. For example, inthe case of liver cell cultures (e.g., for making artificial liverorgans or tissues, or for repairing or reconstructing damaged ordiseased liver), parenchymal liver cells (hepatocytes) are culturedtogether with at least one of the following cells: fibroblast cells,blood vessel cells, Kupffer cells, epithelial cells, endothelial cells,skin cells (keratinocytes), hematopoietic cells, bone marrow cells, etc.Similarly, a culture of other organ cells (e.g., eyes, kidneys, bone,blood vessels, heart) using this exemplary approach can be carried outusing an appropriate combination of cells.

Permanent or Semi-Permanent 3-D Cell/Organ Implants

The invention provides permanent or semi-permanent 2-D and/or 3-Dcell/tissue/organ implants, e.g., for cell, tissue and/or organ growthor reconstruction, or for a medical treatment, e.g., to supplement orreplace a diseased or injured organ or tissue, e.g., a kidney, liver,pancreas, nerve cell(s) (e.g., spinal cord) and the like. An exemplaryartificially engineered nanotube array of the invention comprises a basestructure of wire/rod/ribbon/sheet, as illustrated in FIGS. 19A and 19B,which can be removed, or alternatively, the base structure can beoptionally left in to be a component of inorganic-organic compositestructure. The base structure can be made of Ti wires, ribbons, networksor sponges or any other biocompatible or bio-inert material such as anoble metal, silicon, polymer, etc., optionally coated withbiocompatible or bioinert layer.

In some aspects, for such permanently retained metal structures, alocally bent or curved configuration of the base structure is preferredso as to provide a mechanical locking and more stable structure.

Nanopore-Reservoired Template for Multifunctional 3-D Cell/Organ Growth

The invention provides a nano-reservoir-comprising (e.g., ananopore-reservoired) template for multifunctional 2-D or 3-D cell,tissue and/or organ growth or reconstruction. In one aspect, a benefitof this exemplary artificially engineered nanotube array structure isthat the vertical pores within nanotubes are utilized as a reservoir(e.g., depository) of various biologically active agents to providemulti-functional capability, as illustrated schematically in FIG. 12, orto aid in the growth of, or guide a directional growth of cells, and/orin the maintenance of the cell, tissue and/or organ. In alternativeaspects, therapeutic or test drugs, growth factors, proteins, collagens,enzymes, hormones, nucleic acids (e.g., siRNAs, antisense agents),DNA's, genes, antibiotics, antibodies, magnetic nanoparticles, smallmolecules, lipids, carbohydrates and so forth are used.

The filling of the nanotubes pores (and, in one aspect, also in-betweenthe nanotubes) can be effected by a number of different methods. Forexample, in one aspect, ultrasonic agitation of the nanotube structurein a solution containing one or more of the biologically active agentsis used. In one aspect, nano-sized pores of TiO.sub.2 nanotubes areused. As compared to micro-sized pores, artificially engineered nanotubearray structure of the invention have an advantage of being able to keepthe stored biological agents much longer and allow slower release over alonger period of time; however, arrays and structures of the inventioncan comprise a mixture of micro-sized pores and/or tubes and nano-sizedpores and tubes, depending on the desired reagent release effect.

In one aspect, 2-D and/or 3-D orthopaedic (artificial joint), eye, organ(e.g., liver, kidney), nerve (spinal cord), dental (e.g., odontogenicstructures), skin and/or periodontal (e.g., tooth supporting structures)or any other cell implants of the invention comprise artificiallyengineered nanotube array structures comprising thesenano-reservoir-comprising (e.g., a nanopore-reservoired) templates. Inone aspect, an advantage of building in these nano-reservoirs is that acontinuous supply, a defined duration supply, or an intermittent supplyof biological or chemical agents can be designed in to the compositionof the invention to effect a desired growth, differentiation, cellinteraction or direction cell growth, and the like.

For example, a growth factor can be slowly released from the nanotubes,e.g., a bone morphogenic protein (BMP) type or collagen type growthfactor can be placed into a nano-reservoir (or a micro-reservoir, whichis an alternative option in some aspects). Another example is insertionof infection-preventing antibiotics (such as penicillin, streptomycin,vancomycin, and the like), which can be slowly released from thenanotubes; can be slowly released from the nano-reservoirs. Thus,compositions of the invention can be designed such that much moreefficient and healthier cell or bone growth can be accomplished.

Other examples include 3-D bone growth, 3-D culturing liver or kidney orother organs, 3-D spinal cord, tooth or periodontal tissue re-growth, orre-growth or repair of other cells cells/tissues/organs, which can befurther enhanced by using slowly released biological agents from thenanotube reservoirs. Another example is incorporation of therapeuticagents, e.g., drugs, biological agents or natural products, such asanti-cancer drugs, in the nanotube pores, e.g., where the cultured 3-Dcells/organs are to be implanted into a body of a patient whosecancerous organs have been partially or fully removed.

In addition to the in vitro type cell culture aspects of the invention,the 3-D base structure of the invention can comprise use of artificiallyengineered nanotube array structures pre-filled with one or more typesof drugs and biological agents, where the structures are leftpermanently or semi-permanently in vivo implanted as a source of slowdrug release within an individual, e.g., a human body.

Externally Controllable Drug Release in 3-D Growing Cells/Organs

The invention provides devices, arrays, templates and other structuresof the invention comprising nanotubes and/or nanopores ornano-reservoirs and controlled release compositions, e.g., an externallycontrollable drug release composition, e.g., in a 2-D or 3-D growingcells/tissue/organ of the invention. In one aspect, a device, array,template of the invention comprises multi-functional implants to implantor store remote-controllable media, such as magnetic nanoparticles,biodegradable agents, a colloidal liquid and the like. A biological orchemical agent, such as a cancer drug, can be placed together with (orwithin) a magnetic nanoparticle in the nanopore or nano-reservoir (ormicro-reservoir, which is an alternative option in some aspects), forexample, as in the exemplary nanotubes of FIG. 12.

The nanopore- or nano-reservoir-implanted composition can be designedfor release, for example, by ultrasonic or magnetic agitation of acolloidal liquid containing a mixture of the drug solution and theparticles. Exemplary magnetic nanoparticles for such use includebiocompatible iron-oxide particles of magnetite (Fe.sub.3O.sub.4) ormaghemite (.gamma.-Fe.sub.2O.sub.3), e.g., in the particle size regimeof between about 5 to 50 nm in average diameter. External stimulation ofthe magnetic nanoparticles by orientation-changing magnetic field oralternating current (AC) magnetic field can help release a biologicalreagent, a dye, an isotope (e.g., a radioactive agent), a drug orchemical, e.g., a cancer drug, by mechanical agitation/movement of themagnetic particles or by heating of the drug-particle mixture due to theAC magnetic field.

Magnetic-nanoparticle-containing nanotube structures of the inventioncan also be useful for treatment of a disease, injury or condition(e.g., a cancer) via a combination of externally controllable drugrelease and magnetic hyperthermia treatments, for example, as an implantin bone cancer area or other cancer regions in general. Because therehas been much progress in the general magnetic hyperthermia treatment ofcancer using high frequency alternating current (AC) magnetic field,these treatments can be incorporated when practicing this invention,see, e.g., Jordan (1999) J. of Magnetism and Magnetic Materials194:185-196. The magnetic particles can be confined within thenanotubes, nanopores and/or nano-reservoirs, thus minimizing anycomplications that may arise from the nanoparticles in human body, yetinduce local temperature rise for the magnetic hyperthermia treatment ofcancer.

3-D Cell Proliferation and Supply Device

The invention provides artificially engineered nanotube array structuresthat can accelerate cell growth, e.g., in a 2-D or a 3-D cell growthpattern. For example, the increased number of cells generated by thisexemplary device in a 3-D configuration can be useful for acceleratedsupply of various types of cells, such as bone cells, liver cells,kidney cells, nerve or blood vessel cells, skin cells, periodontalcells, stem cells, and other human or animal organ cells, and otherhuman or animal cells for various R&D or therapeutic uses.

In one aspect, the cells are cultured in a biocompatible environmentwith needed nutrient media. In one aspect, the cells are proliferated onvertically or parallel-aligned nanotube arrays of the invention, andthen can be harvested and supplied for various uses. One exemplarymethod of harvesting the grown 3-D cells off a nanotube arrayedsubstrate comprises use of linearly retractable, base structures; andthen in one aspect, physically or mechanically removing the cells, e.g.,as illustrated in FIG. 16B. Vacuum suction is one of the possibleprocesses for the mechanical removal of the cells from the retractablesubstrate.

Another exemplary method is to use a process known as “trypsinization”.Once cells are grown completely on whole surface of cell culture flask,the media fluid is removed by suction. After rinsing of the cells twicewith PBS (phosphate buffer solution), trypsin is added to detach thecell from the surface. A combination of the template retracting(mechanical removal) and trypsinization can also be utilized. Theretrieved cells are then washed, stored and supplied to the people whowant to purchase them. For trypsinization, generally approximately 2-3ml of trypsin can be used for detaching cells grown on 10 cm.sup.2 cellculture dish. After approximately 2 minutes, most of the cells aredetached from the nanotube surface. After adding approximately 10 ml ofnew medium, this fluid containing the detached stem cells can be pouredinto a centrifuge tube. After centrifugation at appropriate rotationspeed and time, all of the cells can be separated. The medium can beremoved by suction, and 1 ml of new media can be added for storage ofthe harvested cells or for additional culture. To estimate the number ofproliferated cells, trypan blue assay can be employed in conjunctionwith hematocytometrv.

The 2-D or 3-D cultured cells prepared using the artificially engineerednanotube array structures of the invention can be useful for a varietyof applications, e.g., in vivo implanting of cells or organs. Forexample, a patient can be supplied with cultured and implanted,three-dimensional functional cells such as liver, kidney, or bloodvessel cells or organs, as illustrated in FIG. 11B. Three-dimensionallycultured bones or tissues can also be utilized as dental, periodontal ororthopaedic body implants as illustrated in FIG. 11A.

Analytical Diagnostic Biochip

In one aspect, the invention provides analytical diagnostic arrays, orbiochips, comprising structures of the invention. For example, rapidgrowth of cells—such as growth facilitated by a large surface area—canbe effected by use of a composition of the invention. For example, 2-Dor 3-D cell culturing can be useful for carrying out fast diagnosis anddetection of certain types of cells, such as diseases, toxins, poisonsor biological or chemical warfare agents (e.g., bacillus spores, e.g.,anthrax). In one aspect, the invention provides an X-Y matrix subdividedarray, e.g., an artificially engineered nanotube array structureproduced as illustrated in FIG. 17.

For diagnosis of diseases (especially epidemic diseases, e.g., SARS orinfluenza, e.g., the so-called “bird flu”) or biological warfare agents(e.g., bio-terror germs, spores, e.g., anthrax, bacteria or viruses), aswell as identifying cells to produce forensic evidence, the inventionprovides a rapid detection device, even when the available quantity ofcells is relatively small. For example, FIG. 17 illustrates thedetection elements comprising a multiplicity of parallel-configured andlaterally gapped nanotubes of the invention upon which various types ofcells are placed and allowed to rapidly proliferate. In this aspect, asa sufficient number of cells are rapidly grown in a shorter period oftime, an easier and faster detection device and method is generated.

For analysis of cell types, various exemplary techniques, e.g., asillustrated in FIG. 18, can be used, including, e.g., FIG. 18A opticaldetection of morphology and size (using a microscope, fluorescentmicroscope, or CCD camera sensing of fluorescent or quantum dot taggedcells), FIG. 18B chemical or biological detection (e.g., based onsignature reactions), FIG. 18C magnetic sensor detection (e.g., by usingmagnetically targeted antibody and its conjugation with certain types ofantigens).

Liver Cell Array Device for Drug/Chemical Toxicity Testing

Another important application of the compositions of the invention.e.g., the exemplary bio-chip apparatus illustrated in FIG. 17, comprisestheir use as a base structure to culture liver cells for testing of newdrugs, as illustrated in FIG. 24.

A variation of this embodiments includes having a rod or plate shapebase structure in a vertically (or near vertically) arrangedconfiguration; within one aspect, each rod or plate base comprises aparallel—yet a laterally spaced-apart, nanotube array; this can make the2-D or 3-D culture more effective. Because the 2-D or 3-D structured,nanotube array of the invention enables a rapid and healthy culture ofliver cells in a desirable 2-D or 3-D configuration, the apparatus canbe utilized for the important task of testing drug toxicity or chemicaltoxicity.

In one aspect, for toxicity testing, the 2-D or 3-D structuredTiO.sub.2-nanotubes are important for creating a stable “culture”, e.g.,an artificial organ, comprising an array of two- or three-dimensionallyconfigured liver cells. These devices of the invention are importantbecause when a drug is toxic to human or animal body under an in vivosituation, the liver is one of the first organs to sense it and try toisolate the toxic materials. Thus, these devices, e.g., bio-chips, ofthe invention can comprise an array of healthy, 2-D orthree-dimensionally cultured liver cells, e.g., 10.times.10 (10.sup.2),10.sup.3, 100.times.100, 10.sup.5, or 1000.times.1000 cells as sensingelements, thus allowing simultaneous evaluation of many drugs for a muchaccelerated screening and development of biologically acceptable drugs.

Likewise, many chemicals, biological agents, gases, polymers, injectionfluids, and composites and the like that may be useful for in vivoapplications, or may be encountered in in vivo conditions (e.g., toxins,poisons) can be rapidly tested for toxicity, e.g., using the exemplarydevice of FIG. 18 and FIG. 24.

In one aspect, for analysis of the response of liver cells, variousexemplary detection/analysis mechanisms, such as illustrated in FIG. 18can be utilized, for example: 18A optical or microscopic sensing, 18 bchemical or biological detection, and 18C magnetic sensor detection.

III. Articles Comprising Configured Nanotubule Structure,Three-Dimensionally Cultured Cells, Method for Making Such Structure andCulturing Such Cells and Method of Using Such Cells

The invention provides three-dimensionally cultured cells and tissuesgrown by using three-dimensionally configured templates comprisingaligned nanotubule structure. The invention provides three-dimensionallycultured cells/organs, template devices composed of TiO.sub.2 nanotubulestructure which allows a growth of 3-dimensionally configured cells suchas bone cells, liver cells, kidney cells, blood vessel cells, skincells, periodontal cells, stem cells, and methods for applying such cellgrowth techniques. The invention provides vertically aligned TiO.sub.2nanotube arrays adherent on three-dimensionally configured arrays of Tisurfaces; these arrays can induce a strong cell adhesion andsignificantly enhance the formation kinetics of cells. In one aspect,three-dimensionally placed nano-gaps between aligned TiO.sub.2 nanotubesallow a continuous supply of various nutrients to the growing cells. TheTiO.sub.2 nanotube array can be fabricated on the surface of a threedimensional array of wire or ribbon shaped Ti, which can be retractablefrom or permanently kept in the 3-D grown cells. In one aspect, a3-dimensional cell and tissue culture device, with optionally storedbiological agents within the nanotube reservoirs, improves growth ofhealthy liver cells and other cells, and maintains the functionality ofthe cells. Such cultured cells can be useful for rapid supply of neededcells for R&D or therapeutics, for preparation of partial or fullimplant organs, for externally controllable drug release and therapeutictreatments, for efficient toxicity testing of drugs and chemicals, andfor diagnosis/detection of disease or forensic cells.

SUMMARY

The invention provides three-dimensional arrays comprising: a solidsubstrate comprising Ti wires, ribbons or rods, or any combinationthereof, and, a plurality of vertically aligned, laterally spaced,nanotubes associated with the substrate, wherein each nanotube comprisesa nanopore. The outer diameter of each nanotube can be about 10-1000 nm,or about 30-300 nm, or about 60-200 nm.

In one aspect, the nanopore of each nanotube comprises a diameter ofabout 20% of the outer diameter of the nanotube, or, a diameter of about50% of the outer diameter of the nanotube. In one aspect, the height ofeach nanotube is about 40-1000 nm, or, the height of each nanotube isabout 100-400 nm. In one aspect, the aspect ratio of each nanotube isless than about 10, or the aspect ratio of each nanotube is less thanabout 5. In one aspect, the alignment angle of the vertically alignednanotubes is about 0-45 degrees off the vertical direction. In oneaspect, the alignment angle of the vertically aligned nanotubes is about0-30 degrees off the vertical direction. In one aspect, adjacent,vertically aligned nanotubes are laterally spaced from about 2-100 nm,or adjacent, vertically aligned nanotubes are laterally spaced fromabout 5-30 nm.

In one aspect, the nanotubes comprise a biocompatible surface, e.g., acompound comprising Ti, Ti oxide (TiO.sub.2), ceramic, noble metals,and/or polymer materials or a combination thereof. In one aspect, thecompound comprises TiO.sub.2.

The invention provides three-dimensional array compositions and methodsfor three-dimensionally culturing cells, e.g., bone cells, liver cells,kidney cells, blood vessel cells, skin cells, periodontal cells, stemcells, and other human or animal organ cells: wherein the organ cancomprise bone cells, liver cells, kidney cells, blood vessel cells, skincells, periodontal cells, stem cells, and other human or animal organcells.

In one aspect, the three-dimensional array compositions of the inventionfurther comprise a means for retracting the nanotubes from thethree-dimensionally cultured cells such that only the cells/organs areleft. In one aspect, the three-dimensionally cultured cells or organsare permanently or semi-permanently associated with the nanotubes of thearray.

The invention provides compositions and methods for accelerating thegrowth of cells, the method comprising contacting the cells with anarray of the invention (e.g., a three-dimensional array) in the presenceof a nutrient fluid suitable for sustaining growth of the cells.

In one aspect, the cell growth is accelerated by about 100%, 200%, or byabout 300%. In one aspect, the nutrient fluid is supplied under thegrowing cells through the spacing between the parallel nanotubes.

The invention provides compositions (e.g., implants) comprisingthree-dimensional arrays comprising: a solid substrate comprising Tiwires, ribbons or rods, or any combination thereof; and, a plurality ofvertically aligned, laterally spaced, nanotubes associated with thesubstrate, wherein each nanotube comprises a nanopore, and wherein thearray comprises dental cells suitable for implantation and regenerationof dental tissue in a subject.

The invention provides compositions (e.g., implants) comprisingthree-dimensional arrays comprising: a solid substrate comprising Tiwires, ribbons or rods, or any combination thereof; and, a plurality ofvertically aligned, laterally spaced, nanotubes associated with thesubstrate, wherein each nanotube comprises a nanopore and wherein thearray comprises orthopaedic cells suitable for implantation andregeneration of bone and/or joint tissue in a subject.

The invention provides compositions (e.g., implants) comprisingthree-dimensional arrays comprising: a solid substrate comprising Tiwires, ribbons or rods, or any combination thereof; and, a plurality ofvertically aligned, laterally spaced, nanotubes associated with thesubstrate, wherein each nanotube comprises a nanopore, and wherein thearray comprises one or more biologically active agents or chemicalscomprising therapeutic drugs, growth factors, proteins, collagens, stemcells, enzymes, hormones, nucleic acids (e.g., vectors, siRNAs, RNAs,DNAs, genes) antibiotics, antibodies, radioisotopes and/or magneticnanoparticles.

The invention provides compositions (e.g., implants) comprisingthree-dimensional arrays comprising: a solid substrate comprising Tiwires, ribbons or rods, or any combination thereof; and, a plurality ofvertically aligned, laterally spaced, nanotubes associated with thesubstrate, wherein each nanotube comprises a nanopore, and wherein thearray comprises a colloidal composition comprising magneticnanoparticles interspersed with a biological agent selected from thegroup consisting of therapeutic drugs, cancer drugs, growth factors,proteins, collagens, stem cells, enzymes, hormones, nucleic acids (e.g.,vectors, siRNAs, RNAs, DNAs, genes), antibiotics, and antibodies.

The invention provides methods for selectively releasing a biologicalagent in a subject, the method comprising implanting an array of theinventions in a subject and contacting the array with ultrasonic ormagnetic agitation of the colloidal composition, wherein the biologicalagent is released from the array. In one aspect, the magneticnanoparticle comprises iron-oxide particles of magnetite(Fe.sub.3O.sub.4) or maghemite (.gamma.-Fe₂O3) or a combination thereof.The nanoparticles can be about 5-50 m in average diameter. The magneticagitation can comprise external stimulation of the magneticnanoparticles by alternating current (AC) magnetic field. The biologicalagent can be released from the array by mechanical agitation/movement ofthe magnetic particles or by heating of the composition resulting fromthe AC magnetic field.

The invention provides methods for treating a cell proliferationdisorder, the method comprising: implanting a three dimensional array ofthe invention in to a subject, wherein the array is implanted at or nearthe site of a cell proliferation disorder; and, contacting the arraywith magnetic agitation, wherein the agitation accelerates biologicalagent release and provides magnetic hyperthermia treatment at the siteof implantation.

The invention provides systems for growing and harvesting selectedcells, the system comprising: a three dimensional array of the inventionoperably associated with a device for removing the cells or tissue fromthe array; and a computer operably associated with a), wherein thecomputer comprises instructions for automatically contacting the cellswith a suitable growth media and for harvesting the mature cells. In oneaspect, cells are bone cells, kidney cells, blood vessel cells, skincells, liver cells, liver parenchymal cells, endothelial cells,adipocytes, fibroblastic cells, Kupffer cells, kidney cells, bloodvessel cells, periodontal cells, odontoblasts, dentinoblasts,cementoblasts, enameloblasts, odontogenic ectomesenchymal tissue,osteoblasts, osteoclasts, fibroblasts, and other cells and tissuesinvolved in odontogenesis or bone formation, stem cells or a combinationthereof. The cells also can be embryonic or adult stem cells. In oneaspect, the cells are removed by trypsinization. In one aspect, thecells are harvested using mechanical means such as suction andcentrifugal process, or optionally in combination with trypsinization.

The invention provides methods for diagnosing or detecting a condition,disease or an exposure to a toxic agent comprising implanting a biochipcomprising an array (e.g., a three dimensional cell-comprising array) ofthe invention in a subject. The invention provides methods detectingbiological or chemical warfare agents (e.g., anthrax), including gases,bacteria, spores and the like, the method comprising providing an X-Ymatrix subdivided array of nanotube comprising a plurality of nanotubes,each nanotube comprising a specific type of cell for further analysis.

The invention provides biometric or biomimetic arrays comprising livercells (e.g., liver parenchymal cells) for performing drug/chemicaltoxicity testing, the array comprising an array of the invention andliver cells (e.g., liver parenchymal cells) for evaluation of new drugs,cosmetics, dyes, preservatives and/or natural products for testing ofsafety and toxicity issues. In one aspect, the array is contacted with atest material comprising chemicals, polymers or injection fluids. In oneaspect, the “biomimetic” is an “artificially arranged system of livingcells”, or, in addition to conventional material fabrication approachescomprising mimicking the natural biological process (e.g., the devicesand methods of the invention can either be an “artificially arrangedsystem of living cells” or mimic a natural system or process, e.g., atissue or organ system, or a mixture thereof).

The invention provides methods for manufacturing the arrays of theinvention, as described herein.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

DETAILED DESCRIPTION

The invention provides nano-scaled materials that can exhibitextraordinary physical, mechanical and biological properties, whichcannot be achieved by micro-scaled or bulk counterparts.

In one aspect, a TiO.sub.2 phase can be prepared by various techniquessuch as sol-gel method, electrophoretic deposition and anodization. See,e.g., Lakshmi, et al., Chemistry of Materials, Vol. 9, page 2544-2550(1997), Miao, et al., Nano Letters, Vol. 2, No. 7, page 717-720 (2002):Gong, et al., Journal of Materials Research, Vol 16, No 12, page3331-3334 (2001). In one aspect, a TiO.sub.2 phase can be prepared byvarious techniques such as sol-gel method (see, e.g., U.S. Pat. No.7,014,961): photolithographic patterning of a photoresist layer bypattern-wise exposure to short-wavelength ultraviolet light through apattern-bearing photomass, as described in U.S. Pat. No. 6,593,034;electrophoretic deposition and anodization. See, e.g., Lakshmi, et al.(1997) Chemistry of Materials, 9:2544-2550; Miao, et al. (2002) NanoLetters 2:717-720; Gong, et al. (2001) J. Materials Res. 16:3331-3334;Macak (2005) Chem. Int. Ed., 44:7463-7465.

In one aspect, bioactive materials, such as hydroxyapatite and calciumphosphate, are coated on Ti surface; this can make the Ti surface morebioactive. See, e.g., Shirkhanzadeh et al, Journal of Materials ScienceLetters, Vol. 10, page (1991), de Groot et al., Journal of BiomedicalMaterials Research, Vol. 21, page 1375-1381 (1987), and Cotell et al.,Journal of Applied Biomaterials, Vol. 8, page 87-92 (1992). In oneaspect, the interface is bonded with an integrated structure of theinvention comprising a locked-in configuration with a much increasedadhesion area; e.g., a discrete, less continuous layer to minimizeinterface stress and delamination. In one aspect, acceleratedhydroxyapatite and bone growth is accomplished on a mechanicallyadherent TiO.sub.2 nanotube surface on Ti substrate, as described by,e.g., Oh (2005) Biomaterials 26:4938-4943.

The invention provides implants comprising three-dimensionallyconfigured cells, and methods and devices for producing thesethree-dimensionally cultured cells. The invention provides nanotubulestructures useful as a substrate for efficient liver cell culturedevices; and in one aspect, the liver cells are grown quickly in vitrowithout a major loss of their capability for complex functions.

The TiO.sub.2 nanotubule structures of the invention can comprise threedimensional surfaces for cell culture devices in vitro as well as invivo, and bio-artificial liver support devices. This invention provides2-D and 3-dimensional cell culture devices comprising TiO.sub.2nanotubule structures; which in one aspect enable accelerated growth ofcells, e.g., bone cells, kidney cells, blood vessel cells, skin cells,liver cells, liver parenchymal cells, endothelial cells, adipocytes,fibroblastic cells, Kupffer cells, kidney cells, blood vessel cells,periodontal cells, odontoblasts, dentinoblasts, cementoblasts,enameloblasts, odontogenic ectomesenchymal tissue, osteoblasts,osteoclasts, fibroblasts, and other cells and tissues involved inodontogenesis or bone formation, stem cells or a combination thereof;including organized mixed cell groups, including functional organ cellgroups, such as mixed and organized liver or kidney cells: as well asother structural cells such as blood vessel cells, enzyme secretionvessels, periodontal, bone, teeth, or other hard tissue cells. Thesecultured cells can be useful for a variety of applications including i)organ-related therapeutic medical treatment including liver or kidneydisease treatment, ii) orthopaedic, dental or periodontal processes,iii) supply of cells for various research or therapeutic purposes, andiv) disease diagnostic or toxicity testing of new drugs or chemicals.

Referring to the drawings, FIG. 1A-1B schematically illustratesexemplary devices comprising self-organized TiO.sub.2 nanotube arraysgrown on titanium metal or alloy substrate to accelerate cellproliferation according to the invention. In alternative aspects, theTiO.sub.2 nanotubes or any other biocompatible nanotubes desirable forthe exemplary devices have typical desired dimension of about 10-1000 nmin diameter, or about 30-300 nm, or about 60-200 nm in diameter. Inalternative aspects, the desired heights of the tubules are determinedin part by the desired aspect ratio as relatively short height with anaspect ratio of less than about 10, or in one aspect less than 5 ispreferred for ease of storing and eventual dispensing of drugs orbiological agents intentionally placed within the tubule cavity, whichcan maintain the function of the cells being cultured such as livercells, thus help the proliferation of the cells. In alternative aspects,desired height is about 40-1000 nm, or about 100-400 nm.

In alternative aspects, the vertical alignment with open top pore iscrucial for bio implant and related applications being described in thisdisclosure, as the open top of the nanowire allows the penetration ofthe cells into the nanopore cavity for good adhesion as illustrated inFIG. 1, and also allows easy supply of the biological agents stored inthe nanopores. In alternative aspects, the desired angle of verticalalignment is within than 5, 10, 20, 30 or 40 degrees off theperpendicular axis.

In alternative aspects, the base material for the 2D or 3D cell growthstructure can be pure Ti or can be an alloy based on Ti such as Ti—V—Alalloys or other solid solution hardened or precipitation hardened alloyswith increased mechanical strength and durability onto which an oxidenanotubes are formed. In alternative aspects, similar metals such as Zr,Hf, Ce and their oxide nanotubes are also used. Microstructural analysisof the exemplary cell-growth promoting nanostructure was carried outusing by scanning electron microscopy (SEM) and transmission electronmicroscopy (TEM), as depicted in FIGS. 2 and 3. These exemplarystructures comprise vertically aligned, biocompatible TiO.sub.2 with atypical dimension of the hollow nanotubes in FIGS. 2 and 3 beingapproximately 100 nm outer diameter and approximately 70 nm innerdiameter with approximately 15 nm in wall thickness, and approximately250 nm in height.

The exemplary TiO.sub.2 nanotubule array structure shown in FIGS. 2-3was fabricated by anodization technique using a Ti sheet (0.25 mm thick,99.5% purity) which is electrochemically processed in a 0.5% HF solutionat 20 V for 30 min at room temperature. A platinum electrode (thickness:0.1 mm, purity: 99.99%) was used as the cathode. To crystallize theas-deposited, amorphous-structured TiO.sub.2 nanotubes into the desiredanatase phase, the specimens were heat-treated at 500.degree. C, for 2hrs. In alternative aspects, it is preferred that the amorphousTiO.sub.2 nanotubes is crystallized to anatase phase by heat treatment,because an amorphous TiO.sub.2 phase tends to be more susceptible tobreakage by external stresses as compared to a crystalline phase.

In alternative aspects, an important factor for healthy cell growth is acontinuous supply of nutrients including proteins, mineral ions, fluid,etc. to the cell through the flow of body fluid. The gap (spacing)between adjacent exemplary TiO.sub.2 nanotubules in FIGS. 2 and 3 servessuch a function of allowing the body fluid to continuously pass throughand supply nutrients to the bottom side of the growing cells. Inalternative aspects, the desired gap between the nanotubules is in therange of about 2-100 nm, or about 5-30 nm. In some aspects, too small agap reduces the effectiveness of nutrient body fluid flow while toolarge a gap can pose a danger of reduced mechanical stability in theevent of vertical or lateral stress or pressure. A transmission electronmicroscope (TEM) photograph shown for an exemplary inventive TiO.sub.2nanotube array structure, FIG. 3C, gives an average of approximately 15nm spacing between the nanotubes.

Accelerated Cell Growth on TiO.sub.2 Nanotube Array

In order to estimate the effect of having an extremely finenanostructure such as the vertically aligned TiO.sub.2 nanotubes on cellgrowth behavior, an osteoblast cell growth on TiO.sub.2 nanotubes wascarried out as an example. The results indicate that the introduction ofnanostructure significantly improves bioactivity of implant and enhancesosteoblast adhesion and growth. In alternative aspects, an adhesion ofanchorage-dependent cells such as osteoblasts is a crucial prerequisiteto subsequent cell functions such as synthesis of extracellular matrixproteins, and formation of mineral deposits.

All the experimental specimens (0.5.times.0.5 cm.sup.2) used for celladhesion assays were sterilized by autoclaving. A pure Ti sheet polishedby emery paper (#600 grit size) and chemically cleaned was used as acontrol group sample. For cell adhesion studies, MC3T3-E1 osteoblastcells (rat cells of the type CRL-2593, sub-clone 4, ATCC. Rockville,Md.) were used. Each 1 mL of cells was mixed with 10 mL of alpha minimumessential medium (.alpha.-MEM) in the presence of 100/% fetal bovineserum (FBS) and 1% penicillin-streptomycin. The cell suspension wasplated in a cell culture dish and incubated under 37.degree. C., 5%CO.sub.2 environment. When the concentration of the MC3T3-E1osteoblastic cells reached approximately 3.times.10.sup.5 cells/mL, theywere seeded onto the experimental substrate of interest (TiO.sub.2 orTi) which were then placed on a 12-well polystyrene plate, and stored ina CO.sub.2 incubator for 2, 12, 24 or 48 hrs to observe cell morphologyand count viable attached cells as a function of incubation time. Theconcentration of the cells seeded onto the specimen substrate wasapproximately 5.times.10.sup.4 cells/mL.

After the selected incubation period, the samples were washed with 0.1 Mphosphate buffer solution (PBS) and distilled water, respectively, andfixed with 2.5% glutaraldehyde in 0.1 M PBS for 1 hr. After fixing, theywere rinsed three times with 0.1 M PBS for 10 min. For microscopicexamination of cell structures and morphologies, the samples weredehydrated in a graded series of alcohol (50%, 75%, 90% and 100%) for 10min and subsequently dried by supercritical point CO.sub.2. Thedehydrated samples were sputter-coated with approximately 2 nm thickgold for SEM examination. The morphology of TiO.sub.2 nanotubes as wellas that of the adhered cells were observed using SEM and TEM. In thequantitative assay, the adhered cells on sample surface were countedfrom back-scattered SEM images.

In alternative aspects, the invention provides vertically alignedTiO.sub.2 nanotube coatings comprising one or more of the followingstructures, which can provide structural advantages for reducedinterfacial failure:

i) a vertically aligned TiO.sub.2 nanotube coating fabricated to bethin, typically less than 1000 nm, preferably less than 400 nm;

ii) a coating having a strong chemical bonding on the Ti substrate asthe TiO.sub.2 nanotube coating was prepared via chemical process, andsince a common element of Ti is shared by the substrate and the coating.

iii) a TiO.sub.2 nanotube structure of FIGS. 2 and 3 is made to be notcontinuous but is discrete, with a gap between adjacent nanotubes ofapproximately 15 nm. The desired lateral gap dimension is in the rangeof about 2-100 nm, or about 5-30 nm. Such a lateral sub-division of ananotube array structure can be important for minimizing the interfacialstresses between two dissimilar materials joined together, with the twomaterials involved often having substantially different crystalstructure, lattice parameter, and coefficient of thermal expansion.

It has experimentally been confirmed that the vertically alignedTiO.sub.2 nanotubes of the invention are strongly adherent to the Timetal base, as it was very difficult to remove the nanotubes from the Tisurface by attempting to delaminate or scrape off or by bending of theTi substrate. Such a strongly bonded and stable bone-promoting coatingis important, especially in consideration of possible interference byfibroblast cells during bone growth near the Ti implants.

The invention also accommodates the fact that fibroblast cells are proneto attach on smooth surface layers, in contrast to the osteoblasticcells which can attach well on rough surface (see, e.g., Salata (2004)J. Nanobiotechnology 2:3 (2004), and provides such smooth surface layersinterspersed with vertically aligned TiO.sub.2 nanotubes of theinvention. In one aspect, in making a cell-comprising device of theinvention, an opportunity and time is given for the fibrous tissues toform at the boundary interface between the implant and the growing bone.These tissues can keep osteoblasts from adhering onto the surface of aTi implant; however, they can also cause an undesirable loosening of theTi implant. Thus, in another aspect, a rapid and strong adhesion ofosteoblasts on implant surfaces is provided for to effect successfulbone growth.

In addition to the advantages in mechanical properties, the gaps presentbetween adjacent TiO.sub.2 nanotubes of the devices of the inventionalso may be useful as a pathway for continuous supply of the body fluidwith ions, nutrients, proteins, etc. This may contribute positively tothe health of the growing cells. In some aspects, in the absence of suchpathways the proliferating cells may completely cover the bioactiveimplant material surface and the bottom surface of the growingosteoblast cells would then have very limited access to body fluid.

Presented in FIG. 7A-7B are SEM micrographs showing the growth andadhesion of the osteoblast cells (after 2 hrs) on vertically nanoporousTiO.sub.2 nanotubes. The micrographs clearly indicate that the filopodiaof propagating osteoblast cells actually go into the vertical nanoporesof the TiO.sub.2 nanotubes. The observed rapid adherence and spread ofosteoblastic cells cultured on TiO.sub.2 nanotubes could be caused bythree reasons. First, vertically aligned TiO.sub.2 nanotubes exhibitenormously larger surface areas than the flat Ti surface. Second, thepronounced vertical topology contributes to the locked-in cellconfiguration. Thirdly, the pathway in-between TiO.sub.2 nanotube arrayscan allow the passage of body fluid and act as the supply/storage routeof nutrient, which is an essential biological element for cell growth.

FIG. 8A-8C represents the comparative back-scattered SEM micrographs ofthe cells cultured on 8A pure Ti, 8B amorphous TiO.sub.2 nanotubes, and8C anatase TiO.sub.2 nanotubes after 48 hrs of incubation. It is evidentthat the MC3T3-E1 osteoblast cell's adhesion and growth is significantlyaccelerated on TiO.sub.2 nanotubes, and more so on anatase TiO.sub.2nanotubes as compared to the amorphous TiO.sub.2 nanotubes. The plot ofthe number of adhered cells as a function of culture period, FIG. 9,clearly confirms this trend, with the speed of cell adhesion and growthon anatase TiO.sub.2 nanotubes being significantly higher after 48 hrculture, by as much as approximately 400% as compared to the Ti surface.It is noted that at the early stage, e.g., after 2 hr incubation, therewas no significant statistical difference in the data among the threesurfaces investigated However, the number of attached cells on theTiO.sub.2 nanotubes dramatically increases as the culture time isextended to 12, 24 and 48 hrs.

The accelerated cell growth on vertical TiO.sub.2 nanotube array is notrestricted to the osteoblast cells. A similar behavior is seen withother cells, for example, it has been found that liver cell growth issubstantially accelerated on TiO.sub.2 nanotube array. This is shown inFIG. 25 as comparative back scattered electron SEM images of the livercells (rat hepatocyte #AML-12) cultured for 48 hrs on FIG. 25A priorart, sand blasted and roughened Ti using #60 grit sand blasting medium,B inventive TiO.sub.2 nanotube array surface. As is evident from FIG.25B, the liver cells adhere and grow much faster on the verticallyaligned and laterally separated TiO.sub.2 nanotubes than on theprior-art sand blast roughened Ti of FIG. 25A. In fact, not much of theliver cell growth was seen on the sand-blasted Ti, as mostly thedark-colored surface roughness defects covers the SEM image of thesample surface.

Three-Dimensional Cell Growth

The invention provides compositions (e.g., devices, such as artificialorgan systems) for use as organ or hard tissue replacements. In oneaspect, these compositions comprise 2D and/or three-dimensional (3-D)growth of cells. In one aspect, these compositions comprise only 3-Dcell structures, with a substantial volume rather than a thin,two-dimensional (2-D) surface coverage of cells. In this aspect, a 3-Dcell culture technique comprise use of the biocompatible Ti oxidenanotube surface of the invention in a 3-D configuration.

In one aspect, devices of the invention can incorporate cultured cellsgrowing in a two-dimensional configuration covering the surface of acell culture substrate. The invention also provides devices facilitatingcells growing in a three-dimensional configuration, as in the actualliving body; or a combination of 2-D and 3-D configurations.

Three-dimensional cell culturing of some aspects of the inventionprovide for a faster route for growth and supply of increased number ofcells, not only for liver cell related applications, but for producing anumber of other cells in a healthy and accelerated manner. Devices andmethods of the invention can supply, e.g., for implantation, varioustypes of cells, including bone cells, liver cells, kidney cells, bloodvessel cells, skin cells, periodontal cells, stem cells, and other humanor animal organ cells. Devices and methods of the invention can be usedfor drug toxicity testing using toxin-sensitive liver cells, fastdetection and diagnosis of diseased cells and/or possible detection ofbiological warfare agents, including cells exposed to those agents,e.g., including epidemic diseases, anthrax or SARS; and in one aspect,the devices and methods of the invention can utilize only a very smallor trace quantity of cells, e.g., by in one aspect accelerating cellgrowth speed, for example, via the three-dimensional cell culturedevices of the invention.

In one aspect, the invention provides mixed hepatocyte co-culturesystems with non-liver derived cells. In one aspect, this providesmicrobiological environments similar to those in vivo by optimizingcell-cell interactions.

The invention provides a culture method and culture device that willallow artificial in vitro (or in vivo) growth of healthy, fullyfunctional and long-lasting liver cells that can be transplanted to thepatients in need of liver cells. In one aspect, the invention provides aculture method and culture device that can meet the demands for supplyof the cells for toxicity testing of enormous numbers of new orexperimental drugs, chemicals, and therapeutics being developed in thepharmaceutical and chemical industry. With the unique toxin-filteringcapability of liver cells, any toxicity of a new drug can be manifestedfirst by the reaction of the liver cells. In one aspect, the inventionprovides a culture method and culture device comprising an array ofliver cells that can be utilized as a fast testing/screening vehicle tosimultaneously evaluate the potential toxicity of many new drugs andcompounds.

Referring to the drawings, FIG. 23 schematically illustrates anexemplary approach: the growth base is made of subdivided andthree-dimensionally positioned Ti wires, ribbons, or rods, asillustrated in FIG. 23A, which is in this particular case made to beretractable so that the final cultured 3-D cells or organs do notcontain the Ti metal any more as might be desired for some applications,although Ti and TiO.sub.2 are well known to be biocompatible.

The base material can be pure Ti or can be an alloy based on Ti such asTi—V—Al alloys or other solid solution hardened or precipitationhardened alloys with increased mechanical strength and durability. Whileexamples of accelerated cell and bone growth compositions and methods ofthe invention can comprise use of the substrate material comprising Tiand/or its alloys, the compositions (devices) of the invention can alsocomprise other elements having Ti by at least 20%, 30%, 40%, or 500 ormore weight %. Compositions (devices) of the invention can also compriserelated metals such as Zr, Hf, Nb, Ta, Mo, W, and their alloys.Compositions (devices) of the invention can also comprise other metalsand alloys, such as stainless steel, Au, Ag, Pt and their alloys; e.g.,for such 3-D cell growth; and in one aspect, these alternative materialscan be used as long as a coating of Ti and Ti oxide. Zr, Hf, Nb, Ta, Mo,W and/or their oxides, and/or their alloys, with a thickness of at least1, 2, 3, 4, or 5 or more nm, and the coating coverage of at least 20%,30%, 40%, 50%, 60%, 70%, 80% or more of the total surfaces is used.

To obtain relatively large surface area for cell growth, the desiredtitanium metal wire (or rod) diameter or ribbon thickness in FIG. 23A isin the range of about 10.mu.m-50,000.mu.m, or between about25-2,500.mu.m. In one aspect, the desired volume fraction of the metalfor the given targeted cell volume at the end of the planned cellculture period is at least 10% in volume, or at least 30% in volume. Inone aspect, each of the wire or ribbon surfaces is anodized, and can beheat treated to produce aligned; in one aspect, to produce ananatase-structured TiO.sub.2 nanotube array covering the surface. In oneaspect, a preferred dimension of the TiO.sub.2 nanotubes comprises adiameter of between about 10-1000 nm, or between about 30-300 nm, orbetween about 60-200 nm in diameter. In one aspect, a desired heightbeing in the range of about 40-1000 nm, or between about 100-400 nm. Inone aspect, a desired angle is preferably vertical with an allowance of5, 10, 15, 20, 25, 30 or 35 degrees variation off the perpendicularaxis. In one aspect, the desired gap dimension between adjacentnanotubes is in the range of about 2-100 nm, or about 5-30 nm.

In one aspect, for three dimensional cell cultures comprising a mergingof cells grown from adjacent wires or ribbons, a desirable spacingbetween the parallel neighboring branches of wires and ribbons can be atmost 10 times the thickness of an average monolayer cell thickness, oralternatively, at most 5 times the thickness of an average monolayercell. In one aspect, in the case of bone growth, it is the mineral thatgrows—not a living cell, so the desired spacing between the neighboringbranches can be much larger, with a desired spacing being at most 100times the thickness of osteoblast cell, or alternatively, at most 50times.

In one aspect, the shape of these TiO.sub.2 nanotube coated Ti wires orribbons is desirably straight so that the metal arrays can be pulled outafter desired volume of the cells are cultured, as illustrated in FIGS.23B and 23C. The cultured growth will continue after the metal wire orribbon array template structure is pulled out, and any minor surfacedamage or the thin empty gap created by the vacated template will berepaired/filled, as illustrated in the exemplary FIG. 23D. Such athree-dimensionally cultured cell volume in an accelerated andnutrient-supplied manner can be useful for a variety of applicationsincluding creation of a partial or full artificial organs of e.g.,liver, kidney, bone, periodontal tissue, blood vessel cells, skin cells,stem cells, and other human or animal organ cells etc.

The 3-D cultured cells can be in any orientation, as illustrated in FIG.26A-26C. e.g., horizontal, vertical or upside down depending on specificneeds, e.g., the in vivo culture environment, for example, in the caseof organ implants in human, animal, or xenotransplantation of humanorgans which are temporarily cultured in animals prior to humanimplantation.

Growth of Tube-Shaped Cells/Organs

An alternative embodiment of the invention is schematically illustratedin FIG. 27A-27B. Here, an accelerated culture of 3-dimensional,tube-shaped cells (such as blood vessel cells or enzymehormone secretiontubes, intestine tubes) is described. On the Ti wire or rods havingTiO.sub.2 nanotube array surface, the cells are cultured into the tubeconfiguration, after which the rods are pulled out to leave a ready madetubular cells. Such a tube-shaped cells can be used for repairing adamaged blood vessels, enzyme/hormone secretion tubes and intestinetubes.

Co-Culturing

Yet another embodiment of the invention is the 3-dimensional cell growthprocess of culturing at least two types of cells together as illustratedin FIG. 28A-28C. The 3-dimensional cell growth devices of the invention,as the artificial human or animal organs of the invention, can containmore than one type of cells. The invention provides for co-culturing amixed collection of cells (e.g., bone, liver cells), for example, allthe various cell types found in a particular tissue or organ system, oralternatively, enough cell types to sustain an artificial tissue ororgan system to produce a desired effect, e.g., an artificial liver,growth of a tooth, functional pancreas, and the like. Co-culturing amixed collection of cells can be beneficial in obtaining a higherquality cells.

In one aspect, cells are co-cultured using Ti wire or rods havingTiO.sub.2 nanotube array surface, at least two different types of cellscan be co-cultured. For example, in the case of liver cell cultures orartificial livers of the invention, parenchymal liver cells(hepatocytes) are cultured together with at least one of the followingcells: fibroblast cells, blood vessel cells, Kupffer cells, epithelialcells, endothelial cells, skin cells (keratinocytes), hematopoieticcells, bone marrow cells, stem cells, etc. Similarly, a culture of otherorgan cells using the inventive approach can be carried out using anappropriate combination of cells.

In FIG. 28A-28C, an exemplary combination of parenchymal liver cells(hepatocytes) and blood vessel cells is described. After both types ofcells are grown in an appropriate configuration, the Ti wires/rods arraystructure can be pulled out if desired.

Permanent or Semi-Permanent 3-D Cell/Organ Implants

In one aspect of making the 3-dimensional cell growth devices of theinvention, e.g., the artificial human or animal organs of the invention,TiO.sub.2 nanotube-coated Ti wire/rod array structures can be removed.Alternatively, these array structures can be left in to be a componentof inorganic-organic composite structure.

In one aspect, for three dimensional cell cultures comprising a mergingof cells grown from adjacent wires or ribbons, a desirable spacingbetween the parallel neighboring branches of wires and ribbons can be atmost 10 times the thickness of an average monolayer cell thickness, oralternatively, at most 5 times the thickness of an average monolayercell. In one aspect, in the case of bone growth, it is the mineral thatgrows—not a living cell, so the desired spacing between the neighboringbranches can be much larger, with a desired spacing being at most 100times the thickness of osteoblast cell, or alternatively, at most 50times.

Schematically illustrated in FIG. 29A-29D is the configuration of theaccelerated culture of 3-D cells with permanently retained Ti wires,ribbons, networks or sponges having TiO.sub.2 nanotube arrayed surfaces.In one aspect, since both TiO.sub.2 and Ti are known to bebiocompatible, the metal structure does not necessarily have to bepulled out. For such a permanently retained metal structure, a locallybent or curved Ti wire/ribbon configuration is preferred so as toprovide a mechanical locking, more stable structure, as illustrated byFIG. 29A-D.

Nanopore-Reservoired Template for Multifunctional 3-D Cell/Organ Growth10302 In one aspect of making the 3-dimensional cell growth devices ofthe invention, e.g., the artificial human or animal organs of theinvention, a 3-D configured array of Ti wires, rods and ribbons is used.In one aspect, 3-D configured array of Ti wires, rods and ribbons may bebeneficial because the vertical pores of the TiO.sub.2 nanotubes on thesurface of Ti can be utilized as a reservoir of various biologicallyactive agents to provide multi-functional capabilities, e.g., asillustrated schematically in FIG. 13. For example, the nanopores and/ornanotubes of the structures of the invention can be filled, or “loaded”with therapeutic drugs, growth factors, proteins, collagens, enzymes,hormones, nucleic acids (e.g., RNA, DNA, vectors, siRNA, genes),antibiotics, antibodies, magnetic nanoparticles, radioisotopes and soforth. The filling of the TiO.sub.2 nanotubes pores (and also in-betweenthe nanotubes) can be effected by a number of different methods, forexample, using ultrasonic agitation of the TiO.sub.2 nanotube coated Tiin a solution containing one or more of the biologically active agents.The nanosize pores of TiO.sub.2 nanotubes, as compared to microsizedpores, can have an advantage of being able to keep the stored biologicalagents much longer and allow slower release over a longer period oftime. However, the 3-dimensional cell growth devices of the inventioncan also comprise a mix of nanosized pores and micro-sized pores.

In one aspect, 2-D or 3-D organ (e.g., liver, kidney, spinal cord,pancreas), orthopedic (e.g., artificial joints), dental or periodontalcell implants comprising the TiO.sub.2 nanotube coated Ti structure havethe advantage of a continuous supply of biological agents, smallmolecules or other chemicals desired to be used in the cell growth,differentiation or maintenance process. For example, bone morphogenicprotein (BMP) type or collagen type growth factor, orinfection-preventing antibiotics (such as penicillin, streptomycin,vancomycin), or nucleic acids such as vectors, can be slowly releasedfrom the TiO.sub.2 nanotube. An efficient and healthier cell or bonegrowth can be designed.

Similarly as in the case of 3-D bone growth, the invention provides for3-D culturing of other cells or cell systems, such as liver or kidneycells/organs, spinal cord systems, pancreas, and the like, by using theslowly releasing biological agents, small molecules or other chemicalsfrom the TiO.sub.2 nanotube reservoir. Drugs such as anti-cancer drugscan be incorporated in the nanotube pores, e.g., when the cultured 3-Dcells/organ is to be implanted into a body of a patient, e.g., whosecancerous organs have been partially or fully removed.

In addition to the in vitro type cell culture, the 3-D base structure ofTiO.sub.2 nanotube coated Ti (which can be pre-filled with one or moretypes of drugs and biological agents, small molecules or otherchemicals) can also be left permanently or semi-permanently as an invivo implanted 3-D cells/organ as a source of slow drug release withinan individual, e.g., a test or production animal, or a human body,because both Ti and TiO.sub.2 are known to be biocompatible.

Externally Controllable Drug Release in 3-D Growing Cells/Organs

The invention provides multi-functional implants comprisingremote-controllable media, e.g., magnetic nanoparticles. An agent to bedelivered, e.g., a biological agent, such as a cancer drug, or otherreagent, drug, isotope and the like, can be placed, together withmagnetic nanoparticles, in the nanopores of the TiO.sub.2 nanotubes,e.g., as illustrated in FIG. 12. The controllable media, e.g., magneticnanoparticle, can be stimulated/activated to release the substance(e.g., drug, etc.) contained therein, for example, by ultrasonic ormagnetic agitation of a colloidal liquid containing a mixture of thesubstance (e.g., drug solution) and the controllable media, e.g.,particles. Exemplary magnetic nanoparticles for such use includebiocompatible iron-oxide particles of magnetite (Fe.sub.3O.sub.4) ormaghemite (.gamma.-Fe.sub.2O.sub.3) in the particle size regime of about2 to 100 nm, or 5 to 50 nm, in average diameter. External stimulation ofthe magnetic nanoparticles by orientation-changing magnetic field oralternating current (AC) magnetic field can help release the agent to bedelivered. e.g., a biological agent, such as a cancer drug, bymechanical agitation/movement of the magnetic particles or by heating ofthe agent-particle (e.g., drug-particle) mixture due to the AC magneticfield.

In one aspect, magnetic-nanoparticle-comprising TiO.sub.2 nanotubestructures of the invention are useful for treatment of cancer via acombination of externally controllable drug release and magnetichyperthermia treatment, for example, as an implant in bone cancer areaor other cancer regions in general. The methods and compositions of theinvention can incorporate magnetic hyperthermia treatments of cancer,e.g., using high frequency alternating current (AC) magnetic fields,e.g., as described by Jordan (1999) J. Magnetism and Magnetic Materials194:185-196. The magnetic particles can be confined within thenanopores, thus minimizing any complications that may arise from thenanoparticles in human body, yet induce local temperature rise for themagnetic hyperthermia treatment of cancer.

3-D Cell Proliferation-Maintenance and Cell Supply Devices

Cell growth devices of the invention, e.g., the TiO.sub.2 nanotubes ofthe invention, can accelerate 2-D or 3-D cell growth. The increasednumber of cells generated by such a device in a 3-D configuration can beuseful for accelerated supply of various types of cells, such as bonecells, liver cells, kidney cells, blood vessel cells, skin cells,periodontal cells, stem cells, and other human or animal organ cells,and other human or animal cells for various R&D, screening ortherapeutic uses. Cell growth devices of the invention includeartificial organ systems, and systems for growing or regeneratingtissues, e.g., growing teeth or regenerating liver or nerve tissue.Thus, cell growth devices of the invention also incorporate biologicalagents such as biomolecular growth factors, cytokines, collagens,antibiotics, antibodies, drug molecules, small molecules, inorganicnanoparticles, etc. within nanopores, nanotubes or nanoreservoirs formaintenance of these cells.

In one aspect, cells are cultured in or on a device of the invention ina biocompatible environment with needed nutrient media. In one aspect,cells so proliferated on vertically aligned TiO.sub.2 nanotube arrays ofthe invention are then harvested and supplied for other uses. One methodof harvesting the grown 3-D cells off the TiO.sub.2 nanotube substratesis to use a linearly retractable, Ti base structure as illustrated inFIG. 23. Another method is to use a process known as “trypsinization”,discussed above. A combination of the template retracting andtrypsinization can also be utilized; for example, the 3-D cultured cellson an array vertically arranged Ti wires, rods or ribbons (havingTiO.sub.2 nanotube covered surfaces) can be physically removed (e.g., bylow-power vacuum suction) along the vertical direction after appropriatetrypsinization treatment. The retrieved cells are then washed andstored.

In addition to the utility for supply of cells, the 3-D cultured cellscan be prepared using subdivided-Ti based culturing devices of theinvention which comprise TiO.sub.2 nanotubes or nanopores. Harvestedcultured cells, or the devices themselves, can be in vivo implanted ascells, tissues or organs. For example, a patient can be supplied withcultured cells, and/or implanted with a three-dimensional functionalcell system, e.g., functional liver, kidney, or blood vessel cells ororgans, e.g., as illustrated in FIG. 11A. The invention also providesthree-dimensionally cultured bones or tissues can be utilized as dental,periodontal or orthopaedic body implants as illustrated in FIG. 11B.

Analytical Diagnostic Biochip

In one aspect, the rapid growth of cells is facilitated by using a largesurface area on a device of the invention. The invention's 3-D culturingcompositions and methods can be useful for carrying out fast diagnosisand detection of certain types of cells, and certain cell states, e.g.,as in the detection of the presence of a disease or other agent, e.g., apoison or toxin, such as a biological or chemical warfare agent, e.g.,bacillus, such as an anthrax spore.

In one aspect, an X-Y matrix subdivided array of TiO.sub.2 nanotubearray structure can be produced as illustrated in FIG. 17. The devicesof the invention facilitate rapid diagnosis of diseases (especiallyepidemic diseases) or presence of certain agents or chemicals, e.g., apoison or toxin, such as a biological or chemical warfare agent, e.g.,an anthrax spore, bacteria or viruses). The devices of the invention canbe used in the rapid identifying of cells to produce forensic evidence;particularly where rapid detection is essential even when the availablequantity of the cells is relatively small. Each of the exemplarydetection elements in FIG. 24 contains a multiplicity of TiO.sub.2nanotubes on which various types of cells are placed and allowed torapidly proliferate. As a sufficient number of cells are grown in a 3-Dconfiguration in a shorter period of time, this enables an easier andfaster detection.

For analysis of cell types, various exemplary techniques, illustrated inFIG. 18A-18C, can be used, including 18A optical detection of morphologyand size (using a microscope, fluorescent microscope, or CCD camerasensing of fluorescent or quantum dot tagged cells), 18B chemical orbiological detection (e.g., based on signature reactions), 18C magneticsensor detection (e.g., by using magnetically targeted antibody and itsconjugation with certain types of antigens).

Liver Cell Array Device for Drug/Chemical Toxicity Testing

Another application of the bio-chip apparatus of the invention isillustrated in FIG. 17; this exemplary device can be utilized as a basestructure to culture cells, e.g., liver cells, for testing of new drugs,as illustrated in FIG. 24. The device can comprise parenchymal cells(hepatocytes), and/or other cells from a liver environment, such asendothelial cells, adipocytes, fibroblastic cells and Kupffer cells.

The three-dimensionally structured, TiO.sub.2-nanotube-coated array ofTi wires or ribbons enables a rapid and healthy culture of liver cellsin a desirable three-dimensional configuration. In one aspect, theapparatus can be utilized for testing drug toxicity or chemical toxicity(e.g., natural products, perfumes, cosmetics, dyes and the like). Fortoxicity testing, the three-dimensionally structured TiO.sub.2-nanotubescan create a cell culture comprising an array of three-dimensionallyconfigured liver cells, e.g., a mixed cell culture comprising an arrayof three-dimensionally configured liver cells including any combinationof parenchymal cells (hepatocytes), endothelial cells, adipocytes,fibroblastic cells, Kupffer cells.

The invention provides bio-chips comprising an array of healthy,three-dimensionally cultured liver cells, e.g., 10.times.10,100.times.100 or 1000.times.1000 cell sensing elements to allowsimultaneous evaluation of many agents (e.g., poisons, drugs) for muchaccelerated screening and, e.g., development of biologically acceptabledrugs, cosmetics and the like. Any chemical, polymer, injection fluid orcomposite that may be useful for in vivo applications can be rapidlytested for toxicity using a device of the invention, e.g., the exemplarydevices illustrated in FIG. 17 and FIG. 24.

For analysis of the response of liver cells, various exemplarydetection/analysis mechanisms such as illustrated in FIG. 18 can beutilized, for example, 18A optical or microscopic sensing, 18B chemicalor biological detection, and 18C magnetic sensor detection.

It is understood that the above-described embodiments are illustrativeof only a few of the many possible specific embodiments which canrepresent applications of the invention. Numerous and varied otherarrangements can be made by those skilled in the art without departingfrom the spirit and scope of the invention. For example, a thin coatingof biomolecules or chemical molecules can optionally be applied on thesurface of TiO.sub.2 nanotubes to further enhance the attachment ofcells.

For example, the invention provides varied arrangements of laterallygapped nanotube arrays of the invention, and the materials involved,e.g., the nanotubes and the substrate that the nanotubes are adhered to,are not necessarily Ti oxide nanotubes on Ti-based metals, but maycomprise any material, e.g., a biocompatible material. Devices of theinvention can be fabricated using other biocompatible materials ornon-biocompatible materials, e.g., coated with biocompatible and/orbioactive surface layers, such as Ti, or they can be coated with inertbiocompatible surface layers, such as a metal, ceramic or polymer layer.In one aspect, a thin coating of biomolecules or chemical molecules canbe applied on the surface of TiO.sub.2 nanotubes to further enhance theattachment of cells.

In one aspect, nanotube arrays (e.g., for incorporating biologicalagents in a nanopore or nanotube reservoir, and to enhance celladhesion) with desirable gaps between adjacent nanotubes (e.g., to allowcontinuous supply of body fluid and nutrients for healthy cell growth)can be fabricated by using modern high-resolution lithography such aselectron beam lithography, laser beam lithography, ion-beam lithography,or by using self-assembly techniques and associated processingapproaches (see discussion, above).

The examples set forth above are provided to give those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the preferred embodiments of the compositions, and are notintended to limit the scope of what the inventors regard as theirinvention. Modifications of the above-described modes for carrying outthe invention that are obvious to persons of skill in the art areintended to be within the scope of the following claims. All patents andpublications mentioned in the specification are indicative of the levelsof skill of those skilled in the art to which the invention pertains.All references cited in this disclosure are incorporated by reference tothe same extent as if each reference had been incorporated by referencein its entirety individually.

IV. Articles Comprising Dual Structured and Dimension-ControlledBiomaterials Nanostructure for Accelerated Cell and Bone Growth, andMethods for Making Such Structures

The invention provides lock-in nanostructures comprising a plurality ofnanopores or nanotubes, wherein the nanopore or nanotube entrance has asmaller diameter or size than the rest (the interior) of the nanopore ornanotube. The invention provides dual structured biomaterial comprisingmicro or macro pores and nanopores. The invention provides biomaterialshaving a surface comprising a plurality of enlarged diameter nanoporesand/or nanotubes.

SUMMARY

This invention provides novel, biocompatible nanostructuredbiomaterials, devices comprising such biomaterials, and fabricationmethods thereof. The novel biomaterials can enable accelerated cellgrowth and can be useful for a variety of uses including orthopaedic,dental, cell/organ implants, therapeutics, disease diagnostic, drugtoxicity testing, and cell supply applications. The invention provideslock-in nanostructures comprising a plurality of nanopores or nanotubes,wherein the nanopore or nanotube entrance has a smaller diameter or sizethan the rest (the interior) of the nanopore or nanotube. The inventionprovides dual structured biomaterial comprising micro or macro pores andnanopores. The invention provides biomaterials having a surfacecomprising a plurality of enlarged diameter nanopores and/or nanotubes.

The invention provides lock-in nanostructures comprising a plurality ofnanopores or nanotubes, wherein the nanopore or nanotube entrance has asmaller diameter or size than the rest (the interior) of the nanopore ornanotube. The invention provides dual structured biomaterial comprisingmicro or macro pores and nanopores. The invention provides biomaterialshaving a surface comprising a plurality of enlarged diameter nanoporesand/or nanotubes.

The invention also discloses a variety of novel surface configurationsof implant or substrate materials in such a way that not only nanoscaleinterfacial adhesions occur, but microscale and macroscale lock-instructure is also provided to guard against slippage of the implant ontensile stress or breakage of the bond on shear stress. In one aspect,substrate materials comprise novel surface configurations having Ti andTi oxide; and in alternative aspects, also have alloys comprising Ti orTi oxide by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70% or 75% weight %. In anotheraspect, other related materials such as Zr, Hf. Nb, Ta. Mo, W, and theiroxides, or alloys of these metals and oxides by at least 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,60%, 70% or 75% weight % is used. Other materials such as Si, Si oxide,carbon, diamond, noble metals (such as Au, Ag, Pt and their alloys),polymer or plastic materials, or composite metals, ceramics or polymerscan also be utilized. e.g., to produce and use similar desired surfaceconfigurations for bio implant and cell growth applications. In oneaspect, these additional materials (e.g., Si, Si oxide, carbon, diamond,noble metals, etc.) are used; where in one aspect, these material areused as long as a coating of Ti and Ti oxide, Zr, Hf Nb, Ta, Mo, W andtheir oxides, as well as their alloys, are present with a thickness ofat least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nm or more, and the coatingcoverage of at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70% or80% or more of the total surfaces is provided.

The invention describes dimension-controlled biocompatiblenanostructures with accelerated cell growth characteristics as well asenhanced bone growth together with improved adhesion and mechanicalproperties. In one aspect, biomaterials of the invention provide i) adesirable lock-in pore structure with the pore entrance smaller than theremainder of the pore dimension for mechanically stable attachment ofgrown bones or cells, ii) a dual structure comprising macro or microcavities, and iii) sufficiently large nanopores and/or nanotubes bypre-patterning and guided chemical or electrochemical reactions forefficient storage of biological agents and biomolecules for enhancedbio-reactions. In one aspect, these dimension-controlled nanopore and/ornanotube structures either comprise (or consist of) or are covered(completely or partially) with a biocompatible material, such asTiO.sub.2, or equivalent.

In one aspect, the dimension-controlled nanopore and/or nanotubestructures of the invention are useful for rapid production of healthycells including liver cells, bone cells, kidney cells, blood vesselcells, skin cells, periodontal cells, odontoblasts, dentinoblasts,cementoblasts, enameloblasts, odontogenic ectomesenchymal tissue,osteoblasts, osteoclasts, fibroblasts, and other cells and tissuesinvolved in odontogenesis or bone formation and/or stem cells, to namejust a few examples. The structures according to the invention can beuseful for reliable and faster orthopaedic (orthopedic) or dental bonerepair, for preparation of partial or full implant organs, forexternally controllable drug release and therapeutic treatments, forefficient toxicity testing of drugs and chemicals, and fordiagnosis/detection of disease or forensic cells.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

The invention provides nano-scaled materials that, in some aspects, canexhibit extraordinary physical, mechanical and biological propertieswhich cannot be achieved by micro-scaled or bulk counterparts. Theinvention provides compositions comprising Ti and Ti alloys, which arecorrosion resistant, light, yet sufficiently strong for load-bearing,and are machinable. The invention provides compositions comprisingbiocompatible metals which osseo-integrate, either as direct chemical orphysical bonding with adjacent bone surfaces without forming a fibroustissue interface layer.

The invention provides compositions that allow enhanced cell and bonegrowth due to their nanotubule or nanopore design and the Ti orTiO.sub.2 in the nanopore or nanotube configurations. The invention alsoprovides compositions comprising nanostructures made of Ti or TiO.sub.2,or equivalent structures, made of other materials but coated with abiocompatible Ti or TiO.sub.2 film. These structures of the inventionallow enhanced cell adhesion and accelerated growth, for example, by atleast about 10%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 250%,300%, 400% or 500% or more faster.

The invention also provides compositions comprising: lock-innanostructures which have re-entrant characteristics of a nanopore ornanotube entrance, and also having a smaller diameter (or size ingeneral) than the rest of the nanopore or nanotube dimensions on thedevice; in one aspect this allows the cells or bones grown in or on thedevice to be mechanically more firmly attached to the device. In oneaspect, to achieve substantial benefit of the lock-in structure, atleast 10%, 20%, 25%, 50%, 70%, 75%, 80%, 90% or 100% of the pores have alock-in nanostructure. In one aspect, the re-entrant characteristics ofthe nanopore or nanotube entrance comprises having an average entrancediameter (or average pore size if they are not circular) by at least 10%to 50% smaller, including at least 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 70%, 75%, 80%, 90% or 100% smaller than the rest of the nanopore ornanotube dimension so that the cells or bones grown are mechanicallymore firmly attached.

The invention also provides compositions comprising: ii) dual structuredbiomaterials comprising a micro or macro pores in combination withsurfaces covered with finer TiO.sub.2 nanotubes. In one aspect, thedual-sized structures allow micro- or macro-scale growth of bones,essentially completely filling the large pores to guard against theslippage or mechanical failure of grown bones against tensile or shearstresses, while enabling accelerated osteoblast cell growth on thenanotube-covered surface of the implants.

In one aspect, the dual structure comprises micro or macro pores, e.g.,in a re-entrant configuration, and having an average diameter (orequivalent diameter if the pores are not circular) in the range ofbetween about 0.5-1,000.mu.m, or between about 1-100.mu.m, together witha nanostructure consisting of nanopores or nanotubes having an averagepore diameter in the range of between about 30-600 nm. In one aspect,the relative ratio of the micro/macro pores versus (vs) nanopores in thedual structure is such that the micro/macro pores occupy at least 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 70%, 75%, 80%, 90% or more areafraction of the implant or bio-substrate surface; and, in one aspect, atleast 20% but less than 50% to maximize the accelerated cell culture orbone growth via the nanopore or nanotubule portion of the surface.

The invention also provides compositions comprising: iii) enlargeddiameter nanopores and nanotubes suitable for efficient storage ofbiological agents, fabricated by guided chemical or electrochemicaletching. In one aspect, such an enhanced diameter allows easierincorporation of biological or chemical agents such as growth factors,collagens, various proteins/biomolecules, nucleic acids (e.g., vectors,DNA, RNA, siRNA, genes), antibiotics, hormones, drugs such as cancerdrugs or diabetes drugs, radioisotopes, functional particles likemagnetic, metallic, ceramic, polymer particles for hyperthermia ormagnetic hyperthermia treatment of tumors, with the particles optionallyconjugated with other molecules for drug delivery, accelerated cell/bonegrowth, therapeutic treatments, etc. In one aspect, the desirable,enlarged nanopore and nanotube diameter (or equivalent diameter if thepores are not circular) comprises at least about 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800 or 850 or more nm.

In some aspect, biomaterial structures of the invention enableaccelerated growth of cells, e.g., functional organ cells, such as liveror kidney cells as well as other structural cells such as blood vesselcells, enzyme secretion vessels, periodontal cells, odontoblasts,dentinoblasts, cementoblasts, enameloblasts, odontogenicecto-mesenchymal tissue, osteoblasts, osteoclasts, fibroblasts, andother cells and tissues involved in odontogenesis or bone formation, orother hard tissues, and/or stem cells. The availability of such culturedcells can be useful for a variety of applications including i)organ-related therapeutic medical treatment including liver or kidneydisease treatment, ii) orthopaedic, dental or periodontal processes,iii) supply of cells for various research or therapeutic purposes, andiv) disease diagnostic or toxicity testing of new drugs or chemicals.

Nanotube or nanopore structures of the invention can substantiallyimprove cell adhesion and growth kinetics. In one aspect, adhesion ofanchorage-dependent cells such as osteoblasts is a crucial prerequisiteto subsequent cell functions such as synthesis of extracellular matrixproteins, and formation of mineral deposits. In one aspect, many typesof cells beside the osteoblast cells remain healthy and grow fast ifthey are well-adhered onto a substrate surface, while the cells notadhering to the surface tends to stop growing.

The invention provides vertically or parallel-aligned nanostructurearrays, which can have structural advantages for reduced interfacialfailure.

The nanostructure arrays of the invention can comprise vertically orparallel-aligned nanopore or nanotube arrays fabricated to be thin,e.g., less than 2000 nm, or less than 400 nm. The nanostructure arraysof the invention can comprise the same material as a base substratematerial to ensure for strong bonding and mechanical stability of thenanotube or nanopore array, for example, TiO.sub.2-covered nanopores ornanotubes; which in one aspect, have a common element Ti shared with theimplant substrate material titanium to provide a strong chemicalbonding.

Referring to the drawings, FIG. 30A-30E schematically illustratesexemplary configurations of a dimensionally controlled, lock-instructure with the size or diameter of the entrance of the pores madesmaller by oblique incident deposition of biocompatible materials suchas Ti or TiO.sub.2. In one aspect, an oblique incident evaporation orsputtering technique is utilized to deposit a material preferentiallynear the entrance of the nanopores or nanotubes. In one aspect,materials to be deposited as a thin film comprise Ti or TiO.sub.2.

Alloys containing Ti with at least about 10%, 15%, 20%, 25%, 30%, 35%,40% o, 45% or 50% or more weight % Ti can also be utilized. The use ofother related materials such as Zr, Hf. Nb, Ta. Mo, W, and their oxides,or alloys of these metals and oxides by at least about 10%, 15%, 20%,25%, 30%, 35%, 40%, 45% or 50% or more weight % is not prohibited. Othermaterials such as Si. Si oxide, carbon, diamond, noble metals (such asAu, Ag, Pt and their alloys), polymer or plastic materials, or compositemetals, ceramics or polymers can also be utilized to produce and usesimilar surface configurations, e.g., for bio implant and cell growthapplications; and in one aspect comprising a coating of Ti and Ti oxide.Zr, Hf. Nb, Ta, Mo. W and their oxides, as well as their alloys, whereina thickness of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nm andthe coating coverage of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 60%, 70% or 80% more of the total surfaces is provided.

The shadowing effect of the obliquely deposited material, FIG. 30B,results in the lock-in structure with the rotation of the sampleresulting in a more uniform, symmetrical pore entrance narrowing, FIG.30C. The diameter-reducing deposit can also be made on TiO.sub.2nanotubes, FIG. 30D, or on the randomly pored structure prepared forexample, by thin film deposition or etching of a duplex mixed structure.The sputtering for the nanopore entrance narrowing can be carried out byDC, pulse DC or RF sputtering methods. Smooth continuous film, roughtopology film or highly porous structure can be obtained. See, e.g.,Thornton, J. Vac. Sci. Technol. A4(6), 3059 (1986); Meng et al“Investigations of titanium oxide films deposited by dc active magnetronsputtering in different sputtering pressures”, Thin Solid Films 226, 22(1993), by K. Robbie et al “Fabrication of thin films with highly porousmicrostructure”, J. Vacuum Science & Technology, 13(3), 1032 (1995), andJ. Rodriguez et al, “Reactively sputter deposited titanium oxidecoatings with parallel Penniform microstructure”, Adv. Mater. 12(5),341(2000).

Shown in FIG. 31 are various alternative types of lock-in pore structurefor enhanced mechanical stability of grown bones. By virtue of there-entrant nature of the pores, bones grown (and cells cultured) aremechanically better locked, and a tensile stress would not cause thebones or cells partially or fully in the pores would not slip outeasily. FIG. 31A shows exemplary re-entrant oval or circular pores,which can be fabricated by patterned coverage, e.g., by lithographicallypatterned PMMA (polymethylmecarthrylate) or other patternable polymer,followed by isotropic chemical etching of Ti with acids. FIG. 31Brepresents rectangular cavities with corrugating walls, which isfabricated by alternating isotropic vs anisotropic chemical etching orreactive ion etching. Such isotropic vs anisotropic etching techniquesare well known in silicon and MEMS (micro-electro-mechanical systems)fabrication. As an example embodiment, a Ti implant can be isotropic vsanisotropic alternatively etched to obtain a structure of FIG. 31B,which can be oxidized (chemically or by oxygen atmosphere treatment) toexhibit TiO.sub.2 surface. As another example embodiment, a Si wafer canbe isotropic vs anisotropic alternatively etched, to obtain a structureof FIG. 31B, and the surface can be coated by biocompatible Ti orTiO.sub.2 by sputtering or evaporation. FIG. 31C shows re-entranttriangular cross-sectioned pores, which can be fabricated, for example,by anisotropic etching using gradually altered electric field in thecase of electrochemical etching, or gradually increasing concentrationof etchant during the process of pore formation. Shown in FIG. 31D arenanotubes such as TiO.sub.2 with corrugating walls which can befabricated by using alternating larger and smaller electric field duringanodization of Ti and its alloys, for example, by alternating between 15and 20 volts of potential during anodization in hydrofluidic acid orammonium fluoride etchant or by alternating the pH value during theanodization electrochemical etching.

Substrate materials for the exemplary FIG. 31 structures can be Ti or Tioxide as well as alloys containing Ti or Ti oxide by at least 50% weight%. Other related materials such as Zr. Hf. Nb, Ta, Mo, W, and theiroxides, or alloys of these metals and oxides by at least 50% weight %can also be used. Other materials such as Si, Si oxide, carbon, diamond,noble metals (such as Au, Ag, Pt and their alloys), polymer or plasticmaterials, or composite metals, ceramics or polymers can also beutilized to produce and use similar desired surface configurations,e.g., for bio implant and cell growth applications; which in one aspect,these materials can be used as long as a coating of Ti and Ti oxide, Zr,Hf. Nb, Ta, Mo, W and their oxides, as well as their alloys, wherein athickness of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nm and thecoating coverage of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 60%, 70% or 80% more of the total surfaces is provided.

FIG. 32A-32B illustrates titanium nanotubes formed by electrolyticanodization, showing the anodization voltage dependence of TiO.sub.2nanotube diameter. It is seen that anodization of Ti is generallydifficult to provide a large diameter pores or nanotubes with a limitedrange of fabricated TiO.sub.2 nanotube diameter to less thanapproximately 100-150 nm (inside diameter). In some aspects, thisdimension of approximately 100-150 nm is often not large enough toincorporate biological agents and biomolecules, and is also not largeenough for a substantial portion of cells (typically many micrometers insize) to go into the pore and form a locked-in structure, although aportion of the filopodia branches can get into the pore and improve celladhesion and cell growth kinetics.

In some aspects, to obtain a larger pore or nanotube diameter (or alarger average pore entrance dimension), a patterned and guided poreformation, e.g., various lithographical means, is desirable asillustrated in FIG. 33 and FIG. 34. A proper combination of isotropicand anisotropic etching process can be incorporated to form suchstructures. In some aspects, the process can create a “micro-nano dualpore structure” of e.g., in the desired range of about 0.5-1,000.mu.m inaverage diameter, or about 1-100.mu.m diameter range, using the guidedpatterning, the pore surface of which is again covered by e.g., veryfine approximately 100 nm diameter regime TiO.sub.2 nanotubes throughadditional processing of anodization.

The dual structure, illustrated in FIGS. 35A-C as higher magnificationillustrations of the exemplary FIG. 33 and FIG. 34 structures show thedetails of the titanium oxide nanotubes formed on the top and poresurfaces.

In some aspects, such a dual structure is desirable for three purposesof: i) allowing an increased portion of cells into the pores, or forpropagating cells micrometers in size to go into the pores, resulting inenhanced adhesion and accelerated cell growth, ii) enhanced mechanicalstability of grown bone lock-in against tensile or shear stress, and/or,iii) easier insertion/storage of biological agents and biomolecules.

The biological agents that can be stored/trapped in exemplarynanostructures include growth factors, collagens, variousproteins/biomolecules, genes, DNAs, antibiotics, drugs such as cancerdrugs or diabetes drugs, functional particles like magnetic, metallic,ceramic, polymer particles for hyperthermia or magnetic hyperthermiatreatment of tumors, with the particles optionally conjugated with othermolecules for drug delivery, accelerated cell/bone growth, therapeutictreatments, etc. Cell growth inhibiting drugs can also be inserted innanopores or nanotubes of the invention, for example, on the surface ofstents to minimize restenosis or on the surface of other implants (e.g.,drug delivery modules) to prevent/minimize scar tissue formation.

In some aspects, making the dual structured nanopore structure of theexemplary FIGS. 33 to 35, comprises a guided synthesis of“enlarged-diameter nanotubes and nanopores” with essentially verticallyaligned configuration of between about 30, 35, 40 or 45 degrees to about90 degrees angle relative to the surface. Aligned nanopores or nanotubeswith a diameter (or equivalent size) of greater than approximately 150nm or more is difficult to fabricate with currently known methods. Insome aspects, such desired “enlarged-diameter nanotubes and nanopores”can be fabricated by pre-patterning with an array of small craters,e.g., on the order of the diameter of the intended final pore/nanotubediameter first formed on the implant or bio-substrate surface, e.g., bylithography and other approaches as described herein.

The lithographic patterning of craters can be carried out byphotolithography (including UV, deep UV and extreme UV lithography,laser interference lithography, etc.), e-beam or ion beam lithography,nano imprint lithography, and various other known techniques (see alsodiscussion, above). In some aspects, once the pre-patterned craters areformed with a desirable size, location and periodicity, the implant orbio-substrate can be subjected to the chemical, electrochemical or ionetching to form desired larger-diameter aligned pores or nanotubes.

Exemplary structures of the enlarged-diameter nanotubes and nanoporesare schematically illustrated in FIG. 36. The desired range of averagediameter for the enlarged and guided nanopores and nanotubes is at leastapproximately 150 nm, or about at least 200, 250, 300, 350, 400, 450 or500 or more nm. In some aspects, for the sake of desired large surfacearea for accelerated cell and bio reactions, the desired diameter of theenlarged nanopores and nanotubes is kept below 0.5, 1, 2, 3, 4, 5, 6, 7,8, 9, or 10.mu.m; unless, in one aspect, a dual micro-nano structure ofthe invention is introduced to make all exposed surfaces of the enlargedpores or nanotubes covered with yet finer TiO.sub.2 nano nanotubes.

Described in FIGS. 36 to 40 are various exemplary techniques forobtaining enlarged-diameter nanotube and nanopore structures. Shown inFIG. 36 is an exemplary lithographic patterning approach for craterarray fabrication, while FIG. 37A-37C schematically illustrates anexemplary process of using nano imprinting to pre-pattern craters on Tior TiO.sub.2 surface for guided synthesis of larger diameter TiO.sub.2nanotubes and nanopores. Such nano imprint lithography technique isconvenient for mass production of a large number of implants or biosubstrates as simple stamping operation can accomplish thepre-patterning of craters without resorting to complicated and oftencostly photolithography or e-beam lithography patterning of each part.In some aspects, another major advantage is the ability to useelastomeric stamp with compliance, which allows reliable stamping onlarge-area, nominally-flat surfaces of Ti or other biocompatiblesubstrates and implants in which the flatness is not always guaranteedto ensure reliable stamping over a large sample area.

In some aspects, the bio implant parts are more often than not in anon-flat surface geometry. Therefore it is desirable to have aconvenient and effective means of fabricating the pre-patterned cratersfor guided introduction of enlarged-diameter nanopores or nanotubes,e.g., on the surface of Ti implants. FIG. 38A-38B illustrates exemplaryprocesses of guided etch nano patterning of identical patterns onnon-flat surfaces using 38A conformable or stretchable elastomeric masksheet, 38B elastomeric roll stamping.

In some aspects, a technique of forming such a desired uniform nanoporeor nanotube array on non-flat surface comprises introducing guidedetching using a vertically two-phase decomposable coating as illustratedin FIG. 39A-39F. Here, a non-flat substrate or implant (e.g., bent Tiwire) is coated with a material which is then decomposed into avertically aligned two-phase structure (e.g., decomposable diblockcopolymer layer heated to decompose into two phases, a spinodal alloycoating heated to decompose into two phases, etc.) as illustrated inFIG. 39A-B. The two phase structure is then differentially etched, e.g.,by chemical etching or ion etching to exhibit a nanopored structure asillustrated in FIG. 39C. The base non-flat substrate or implant materialis etched through the nanopores in the coating layer to create an arrayof craters (FIG. 39D). The coating layer material is then etched away(FIG. 39E) and the substrate is subjected to anodization or chemicaletch to introduce deeper nanopores or nanotubes on the implant surface(FIG. 39F).

In some aspects, diblock copolymers are made up of two chemicallydifferent polymer chains or blocks while they are joined by a covalentbond. Because of this connectivity constraint yet chemicalincompatibility with each other, the diblock copolymers tend to phaseseparate and self-assemble into an ordered (often with a hexagonalgeometry), nanoscale, mixed-phase composites. Depending on the chemistryand decomposition conditions, they can form an ordered array with one ofthe polymer components taking a nano-cylinder shape embedded in theother polymer component. Examples of diblock copolymers include amixture of polystyrene-polybutadiene and that ofpolystyrene-polyisoprene. The diblock copolymers are diluted with asolvent such as toluene, and can be dip coated, brush coated or spraycoated on a substrate. When the heat is applied and drying proceeds andthe copolymer concentration and temperature reaches a critical point,the phase decomposition of the diblock copolymer into an orderedstructure takes place. The desired temperature rise to nucleate and growthe ordered decomposed diblock copolymer structure is typically in therange of between about 50.degree. C. to 35.degree. C., or between about100.degree. C. to 250.degree. C.

In some aspects, the spinodal alloys can be spontaneously decomposedinto a uniform two phase structure by heating to a high temperaturewithin the spinodal range. Fe—Cr—Co, Al—Ni—Co—Fe, Cu—Ni—Fe, Cu—Ni—Co,and Al—Si alloys are well known examples of spinodal alloys. Due to thedifference in chemical etchability between the two decomposed phases, ananoporous structure of FIG. 39C can be obtained.

FIG. 40A-40E illustrates an exemplary size-controlled, uniform nanoporeor nanotube array on various surfaces by guided etching using avertically two-phase decomposed coating of periodically decomposingdiblock copolymer or spinodally decomposing alloy of FIG. 39. Thesize-controlled, uniform nanopore or nanotube array can be formed onvarious exemplary surfaces by guided etching, i.e., on a flat surface(FIG. 40A), on a coarse-patterned surface (FIG. 40B), on parallel Tisheet or wire array (FIG. 40C) which can be useful for three-dimensionalcell or organ structure, on wire mesh, wire bundle or scaffold typehighly porous metal foam (e.g., made of Ti or its alloy) (FIG. 40D), andon re-entrant cavity surface (e.g., pre-shaped Ti or Ti-coated Si) (FIG.40E).

In some aspects, the 3-D cultured cells are prepared using parallel Tisheet, wire array or foam arrays with nanopore or nanotube array surfacestructure, e.g., see the exemplary structures in FIGS. 40C and D, thesecan be useful for a variety of applications including in vivo implantingof cells or organs. A patient can be supplied with in vitro cultured andin vivo implanted, three-dimensional functional cells such as liver,kidney, or blood vessel cells or organs. Three-dimensionally culturedbones or tissues can also be utilized as dental, periodontal ororthopaedic body implants.

In some aspects, to obtain relatively large surface area for cellgrowth, the desired thickness of the titanium metal ribbon, or thedesired diameter of the metal wire or foam in FIG. 40C or (d) is in therange of about 10.mu.m-50,000.mu.m, or about 25 to 2,500.mu.m. In someaspects, the desired volume fraction of the metal for the given targetedcell volume at the end of the planned cell culture period is at least10% in volume, or at least 30% in volume. In some aspects, each of theribbon surfaces is made to contain an aligned nanopore or nanotubearray; in some aspects, with a diameter being in the range of about10-1000 nm, or about 30-300 nm, or about 60 to 200 nm in diameter; insome aspects, the desired height being in the range of about 40 to 2000nm, or about 100-400 nm; in some aspects, the desired angle is verticalwith an allowance of about 10, 20, 30, 40 or 50 degrees variation offthe perpendicular axis.

In some aspects, the shape of these nanostructure-surface ribbons isstraight so that the metal arrays can be optionally pulled out afterdesired volume of the cells are cultured. In some aspects, the culturedgrowth will continue after the metal wire or ribbon array templatestructure is pulled out, and any minor surface damage or the thin emptygap created by the vacated template will be repaired/filled by growingcells. In some aspects, such a three-dimensionally cultured cell volumein an accelerated manner can be useful for a variety of applicationsincluding creation of a partial or full artificial organs of e.g.,liver, kidney, bone, periodontal tissue, blood vessel cells, skin cells,stem cells, and other human or animal organ cells etc. In some aspects,the 3-D cultured cells, according to the invention, can be in anyorientation, i.e., horizontal, vertical or upside down depending onspecific needs especially in the in vivo culture environment, forexample, in the case of organ implants in human, animal, orxenotransplantation of human organs which are temporarily cultured inanimals prior to human implantation.

In some aspects, biological agents or functional nanoparticles canadditionally be stored in the surface nanopores or nanotubes on thesevarious shaped substrates or implants. The functional nanoparticlesinclude magnetic particles, novel metal or SPR (surface plasmonresonance) based photoluminescent particles, quantum dots, fluorescenceparticles, bio-conjugated particles, which are added for the purpose ofaccelerated cell/bone growth, protein harvest (as a result ofaccelerated cell growth, proliferation, and secretion), delivery ofdrugs, genes, chemicals, therapeutics, etc.

Referring to the drawings, FIG. 41A-41D schematically illustrates anexemplary bone- or cell-locking nanopore/nanotube array with biologicalagents inserted for accelerated bone or cell growth, protein harvest,drug delivery, and therapeutics. As described earlier in relation toFIG. 30A-30E, dimensionally controlled, lock-in structures with the sizeor diameter of the entrance of the pores made smaller than the rest ofthe pore dimension below by oblique incident deposition of biocompatiblematerials such as Ti or TiO.sub.2. An oblique incident evaporation orsputtering technique is utilized to deposit a material preferentiallynear the entrance of the nanopores or nanotubes.

In some aspects, the biological agents that can be stored/trapped innanostructures of the invention comprise growth factors, collagens,various proteins/biomolecules, genes, DNAs, antibiotics, hormones, drugssuch as cancer drugs or diabetes drugs, functional particles likemagnetic, metallic, ceramic, polymer particles for hyperthermia ormagnetic hyperthermia treatment of tumors, with the particles optionallyconjugated with other molecules for drug delivery, accelerated cell/bonegrowth, therapeutic treatments, etc.

Cell growth inhibiting drugs can also be inserted in the nanopores ornanotubes of the invention, for example, on the surface of stents tominimize restenosis or on the surface of other implants (e.g., drugdelivery modules) to prevent/minimize scar tissue formation. Theentrance-diameter-reducing deposit can be made on a variety of porousstructures such as on rectangular cross-section shape pores, onre-entrant shape pores, on TiO.sub.2 nanotubes, or on the randomly poredstructures prepared for example, by thin film deposition or etching of aduplex mixed structure. FIG. 12A-12D schematically illustrates cells orbone growing and locked in on these entrance-diameter-reduced orre-entrant shaped nanostructure surface.

In some aspects, instead of biomolecules, functional nanoparticles suchas magnetic particles, novel metal or SPR (surface plasmon resonance)photoluminescent particles, quantum dots, fluorescence particles,bio-conjugated particles for delivery of drugs, genes, chemicals, etc.can be stored in the pores on various shaped nanostructure surface asillustrated in FIG. 43A-43D, for accelerated cell or bone growth,protein harvest, drug delivery, and therapeutics such as hyperthermia ormagnetic hyperthermia treatment of tumors.

FIG. 11A-11C schematically illustrates various potential in vivo or exvivo applications of exemplary dimension-controlled biomaterials capableof accelerated cell/bone growth or functional drug delivery andtherapeutics. In some aspects, nanopore or nanotube structures describedin relation to FIG. 30A to 43D can be utilized for these biomedicaldevice applications. Examples shown include orthopaedic and dentalimplants, cell or organ implants, drug delivery devices such ascontrolled release of insulin by magnetic actuation, artificial liverdevices, drug-protected stents (e.g., to prevent/minimize restenosis) orother tubules inserted into blood vessels and in various other bodyparts, and therapeutic devices such as magnetic field induced localheating for cancer treatment. In some aspects, cell growth inhibitingdrugs can be inserted in the exemplary nanopores or nanotubes ofimplants such as drug delivery modules to prevent/minimize scar tissueformation.

Illustrated in FIG. 15 is an exemplary magnetically actuated, on-offcontrollable or programmable, remote drug release device comprisingexemplary biomaterials of the invention. Remote magnetic field such asapproximately 100 KHz AC magnetic field can be utilized to activate thedrug release by preferential heating of the magnetic nanoparticlesstored in the nanopores of implants, thus allowingtemperature-gradient-induced drug movement from the nanopores into thehuman body. Suitable magnetic nanoparticles for such use includebiocompatible iron-oxide particles of magnetite (Fe.sub.3O.sub.4) ormaghemite (.gamma.-Fe.sub.2O.sub.3) in the particle size regime of about5 to 50 m in average diameter.

In some aspects, a mechanical agitation and induced movement of themagnetic particles themselves in the pores containing the stored drugscan be utilized as an alternative mechanism of drug release. In thiscase, a rather lower frequency AC magnetic field actuation with agradient magnetic field is preferred in order to allow movement ofmagnetic particles against the viscosity of the fluid in the nanopores.In some aspects, a desired frequency range is approximately 1-10,000 Hz,or between about 10-1,000 Hz.

In these exemplary approaches, the drugs are released any time of theday only when desired, and can be turned off completely when it is nolonger needed, so the side effects of drugs are minimized. In someaspects, similar magnetic particle and external field combination canalso be utilized for cancer treatment, for example, through an implantdevices containing the nanoporous structure (e.g.,entrance-diameter-reduced nanopores or nanotubes) placed in the vicinityof cancerous regions, for example, in the liver containing inoperabledistribution of recurring/growing cancer cells. In some aspects,repeated hyperthermia treatments, e.g., over many months, are used,e.g., by on-off type continual activation of external magnetic field cangradually kill the cancer cells near the cancerous regions.

FIG. 16A-16B illustrates an exemplary method of harvesting cellscultured in an accelerated way using dimension-controlled biomaterials.In some aspects, the nanopore or nanotube structures comprisingTiO.sub.2 or related materials accelerate growth of certain types ofcells. In some aspects, the increased number of cells generated by sucha device can be useful for accelerated supply of cells, especially rarecells such as stem cells for various R&D or therapeutic uses. Asillustrated in FIG. 16A, the cells are cultured in an exemplarybiocompatible environment with needed nutrient media. The cells soproliferated on the nanopore or nanotube arrays are then harvested andsupplied for other uses.

One method of harvesting the grown cells off the biomaterial substrateis to use a process known as “trypsinization”. Once cells are growncompletely on whole surface of cell culture flask, the media fluid isremoved by suction. After rinsing of the cells twice with PBS (phosphatebuffer solution), trypsin is added to detach the cell from the surface.In general, approximately 2-3 ml of trypsin is used for detaching cellsgrown on 10 cm.sup.2 cell culture dish. After a few minutes, most of thecells are detached from the surface, as illustrated in FIG. 16B. Afteradding approximately 10 ml of new medium, this fluid containing thedetached stem cells is poured into a centrifuge tube. Aftercentrifugation at appropriate rotation speed and time, all of the cellsare separated. The medium is removed by suction, and approximately 1 mlof new media is added for storage of the harvested cells or foradditional culture. To estimate the number of proliferated cells, trypanblue assay are employed in conjunction with hematocytometry. The cellscan also be in situ proliferated on the implant surfaces as theTiO.sub.2 as well as Ti substrate are biocompatible.

In some aspects, the dimension controlled Ti oxide nanopore or nanotubestructure is also useful for carrying out fast diagnosis and detectionof certain types of cells such as diseased cells, or cells exposed tobiological or chemical warfare agents. In some aspects, an X-Y matrixsubdivided array of dimension-controlled nanopore or nanotube arraystructures are produced as illustrated in FIG. 44. Various exemplarynanopore or nanotube structures described in relation to FIGS. 30 to 43,can be utilized for accelerated cell detection/diagnosis.

In some aspects, the dimension controlled Ti oxide nanopore or nanotubestructures are used in the diagnosis of diseases (especially epidemicdiseases) or detection of toxins, bacteria or viruses, e.g., where arapid detection is essential even when the available quantity of thecells is relatively small. Each of the detection elements in FIG. 44which contains a multiplicity of the dimension controlled nanopores ornanotubes on which various types of cells to be analyzed are placed andallowed to rapidly proliferate to a sufficient number for easydetection.

For analysis of cell types, various exemplary techniques, illustrated inFIG. 18A-18C can be used, including 18A optical detection of morphologyand size (using a microscope, fluorescent microscope, or CCD camerasensing of fluorescent or quantum dot tagged cells), 18B chemical orbiological detection (e.g., based on signature reactions), 18C magneticsensor detection (e.g., by using magnetically targeted antibody and itsconjugation with certain types of antigens).

Another exemplary application of the bio-chip apparatus of the inventionis illustrated in FIG. 44; this aspect utilizes the bio-chip apparatus abase structure to culture liver cells for testing of new drugs, asillustrated in FIG. 45, showing an exemplary bio-chip device fortoxicity testing of drugs or chemicals, with the device comprising anarray of cultured liver cells grown rapidly and healthy manner on thedimension-controlled biomaterials. As in the cell detection/diagnosisapplications, various the bio-chip apparatus nanopore or nanotubestructures, e.g., as described in FIGS. 30 to 43, can be utilized forthe liver cell culture and toxicity test applications. A variation ofthe embodiments include having a parallel wire, rod or plate shape basestructure in a vertically (or near vertically) arranged configuration(with each rod or plate base containing parallel, yet laterally spacedapart nanopore or nanotube array) in order to make the 3-D culture moreeffective.

In some aspects, the three-dimensionally structured, nanopore ornanotube arrays of the invention enable a rapid and healthy culture ofcells, e.g., liver, kidney or other cells, in a desirablethree-dimensional configuration. In some aspects, the apparatus isutilized for testing drug toxicity or chemical toxicity. The inventionprovides bio-chips comprising arrays of healthy, three-dimensionallycultured liver cells, e.g., 10.times.10, 100.times.100, or1000.times.1000 cells as sensing elements to allow simultaneousevaluation of many drugs for much accelerated screening and developmentof biologically acceptable drugs. Likewise, many chemicals, polymers,injection fluids, and composites that may be useful for in vivoapplications can be rapidly tested for toxicity using the exemplarydevice of FIG. 19A-19C. The accelerated, healthy liver cell growthdevice of FIG. 19A-19C can also be utilized as the basis of artificialliver devices for patients waiting for transplant or as a temporary aidto liver function after transplant. Other functional organs applicationssuch as artificial kidney can also be considered.

It is understood that the above-described embodiments are illustrativeof only a few of the many possible specific embodiments which canrepresent applications of the invention. Numerous and varied otherarrangements can be made by those skilled in the art without departingfrom the spirit and scope of the invention. For example, the materialsinvolved do not have to be Ti oxide nanotubes on Ti-based metals, as thenanotubes and the substrate that the nanotubes are adhered to can beother biocompatible materials or non-biocompatible materials coated withbiocompatible and bioactive surface layer such as Ti, or coated withbiocompatible but bio-inert surface layer such as novel metal or polymerlayer. Also, a thin coating of biomolecules or chemical molecules canoptionally be applied on the surface of TiO.sub.2 nanopores or nanotubesto further enhance the attachment of cells.

V. Biomaterials with Size-Randomized Surface Nanopores, FabricationMethod Thereof, and Devices and Articles of Manufacture Comprising SuchMaterials

The invention provides products of manufacture comprising asize-randomized and shape-randomized nanopore- or nanotube-comprisingsurfaces. In one aspect, these compositions are made by a methodcomprising providing a composition comprising a Ti or Ti oxide surface,depositing a semi-wettable coating on the Ti or Ti oxide surface byemploying lithographic patterning or by a thin film depositiontechnique, wherein the coating decomposes into nano- or micro-islands oflocal etch masks; and chemical etching or electrochemical anodization ofthe etch-masked surface, thereby generating a size-randomized andshape-randomized nanopore- or nanotube-comprising surface.

In some aspects, because the same type of cells having substantiallyvarying cell size and shape from one cell to another, e.g., liver cells,grow better when co-cultured with other types of cells, such asendothelial or fibroblast cells which have significantly different cellsizes, the invention provides devices and methods comprising use ofmixed cell cultures. e.g., liver cells, pancreas cells and the like,with endothelial cells, fibroblast cells, fat cells, blood vessel cellsstem cells and the like. Aspects of the invention comprisingsize-randomized nanopores and nanotubes allow efficient culture andco-culturing of cells, for example, for artificial in vitro or in vivogrowth of healthy, fully functional and long-lasting liver cells.

SUMMARY

The invention provides novel, biocompatible nanostructured biomaterials,devices comprising such biomaterials, and fabrication methods thereof.The novel biomaterials can enable accelerated cell growth and can beuseful for a variety of uses including orthopaedic, dental, cell/organimplants, therapeutics, disease diagnostic, drug toxicity testing, andcell supply applications. The invention provides products of manufacturecomprising a size-randomized and shape-randomized nanopore- ornanotube-comprising surfaces. In one aspect, these compositions are madeby a method comprising providing a composition comprising a Ti or Tioxide surface; depositing a semi-wettable coating on the Ti or Ti oxidesurface by employing lithographic patterning or by a thin filmdeposition technique, wherein the coating decomposes into nano- ormicro-islands of local etch masks; and chemical etching orelectrochemical anodization of the etch-masked surface, therebygenerating a size-randomized and shape-randomized nanopore- ornanotube-comprising surface.

In some aspects, preferred substrate materials with exemplary surfaceconfigurations comprise Ti and Ti oxide, and/or alloys containing Ti orTi oxide by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%or more weight %. However, the use of other related materials such asZr, Hf. Nb, Ta, Mo, W, and their oxides, or alloys of these metals andoxides by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%or more weight % can be used. Other materials such as Si, Si oxide,carbon, diamond, noble metals (such as Au, Ag, Pt and their alloys),polymer or plastic materials, or composite metals, ceramics or polymerscan also be utilized to produce and use similar desired surfaceconfigurations, e.g., for bio implant and cell growth applications;where in one aspect, these materials are used as long as a coating of Tiand Ti oxide, Zr, Hf; Nb, Ta, Mo, W and their oxides, as well as theiralloys, with a thickness of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ormore nm and the coating coverage of at least 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 60%, 70% or 80% more of the total surfaces isprovided.

The invention provides size-randomized biocompatible nanostructures withaccelerated cell growth characteristics as well as enhanced bone growthtogether with improved adhesion and mechanical properties. The exemplarybiomaterials have a range of nanopore or nanotube diameters which allowmore efficient co-culture of various cells, in particular, for efficientculture of healthy liver cells and organs as a mixed co-culture ofhepatocyte cells together with non-hepatocyte cells. Suchsize-randomized structures with biocompatible surface are fabricated,according to the invention, by utilizing semi-wettable coating thatdecomposes into nano or micro islands of local etch masks, by employinglithographic patterning, or by thin film deposition technique, followedby chemical etching or electrochemical anodization of Ti or relatedmaterials in various configurations for planar and three-dimensionalcell or bone culture. Such biocompatible nanostructures can be usefulfor reliable and faster orthopaedic or dental bone repair, forpreparation of partial or full implant organs or artificial ex-vivodevices such as artificial liver devices, for externally controllabledrug release and therapeutic treatments, for efficient toxicity testingof drugs and chemicals, and for diagnosis/detection of disease orforensic cells.

The invention provides products of manufacture comprising asize-randomized and shape-randomized nanopore- or nanotube-comprisingsurface made by a method comprising the following steps: (a) providing acomposition comprising a Ti or Ti oxide surface; (b) depositing asemi-wettable coating on the Ti or Ti oxide surface by employinglithographic patterning or by a thin film deposition technique, whereinthe coating decomposes into nano- or micro-islands of local etch masks;and (c) chemical etching or electrochemical anodization of theetch-masked surface, thereby generating a size-randomized andshape-randomized nanopore- or nanotube-comprising surface.

In one aspect, wherein the composition comprising a Ti or Ti oxidesurface comprises a Ti- or Ti oxide-comprising plate, sheet, wire, meshor foam. In one aspect, the distribution of the nanopore or nanotubesizes is such that at least one third of the pores or tubes have theiraverage diameter equal to or less than 30%, 40%, 50%, 60% or 70% of theoverall average pore diameter, while another one third of the pores havetheir average diameter at least 100%, 125% or 150%, the overall averagepore diameter. In one aspect, the at least one third of the pores ortubes have their average diameter equal to or less than 10%, 20%, 30%,40%, 50% or 60% of the overall average pore diameter. In one aspect, theat least one quarter of the pores or tubes, or 10%, 20%, 30%, 40%, 50%or 60% of the pores or tubes, have their average diameter equal to orless than 10%, 20%, 30%, 40%, 50% or 60% of the overall average porediameter. In one aspect, the one third of the pores have their averagediameter at least 115%, 125%, 150%, 175%, 200% or more of the overallaverage pore diameter.

In one aspect, the nanopore or nanotube entrance has a smaller diameteror size than the rest (the interior) of the nanopore or nanotube. In oneaspect, the nanopore or nanotube entrance has an average entrancediameter or average pore size by at least 10% to 50% smaller, or atleast 15%, 20%, 25%, 30%, 35%, 40% or 45%, smaller than the rest (theinterior) of the nanopore or nanotube dimension.

In one aspect, the nanopore or nanotube have an average diameter, orequivalent diameter if the pores are not circular, in the range ofbetween about 0.5 to 1.000.mu.m, or between about 1 to 100.mu.m, andoptionally the entrances of the micro or macro pores have a smallerdiameter or size than the rest (the interior) of the micro or macropores; and, a surface area covered with TiO.sub.2 nanotubes having anaverage pore diameter in the range of between about 30-600 mm, whereinoptionally the relative ratio of the micro/macro pores of (a) and thenanopores of (b) in the dual structure is such that the micro/macropores occupy at least 10% area fraction of the dual structuredbiomaterial, or optionally at least 20% but less than 50% o.

In one aspect, the product of manufacture further comprises a pluralityof cells, wherein the cells comprise liver cells, liver parenchymalcells, endothelial cells, adipocytes, fibroblastic cells, Kupffer cells,kidney cells, blood vessel cells, skin cells, periodontal cells,odontoblasts, dentinoblasts, cementoblasts, enameloblasts, odontogenicectomesenchymal tissue, osteoblasts, osteoclasts, fibroblasts, and othercells and tissues involved in odontogenesis or bone formation, stemcells or a combination thereof.

The invention provides products of manufacture comprising any of thecompositions of the invention described herein comprising Ti and Tioxide; alloys comprising Ti or Ti oxide by at least 50% weight %; Zr;Hf; Nb; Ta; Mo; W; oxides of Zr, Hf; Nb, Ta. Mo or W; or alloys of Zr,Hf; Nb, Ta, Mo or W comprising at least 50% weight % of Zr, Hf. Nb, Ta,Mo or W; or Si, Si oxide, carbon, diamond, noble metals, Au, Ag, Pt, orAu, Ag or Pt alloys, polymer or plastic materials, or composite metals,ceramics or polymers with a coating of biocompatible Ti and Ti oxide,Zr, Hf; Nb, Ta. Mo, W and their oxides or alloys of Zr, Hf; Nb, Ta, Mo,W; and optionally with a thickness of at least about 1, 2, 3, 4 or 5 ormore nm; and optionally the coating coverage of at least about 20%, 30%,40%, 50%, 60%, 70%, 80% or more of the total surfaces.

In one aspect, the product of manufacture further comprises a biologicalagent, small molecule, or other composition, e.g., a growth factor, acollagen, a nucleic acid, an antibiotic, a hormone, a drug, a magneticparticle, a metallic particle, a radioisotope, a ceramic particle, apolymer particle, a drug delivery particle, a lipid, a liposome, acarbohydrate, a nucleic acid or a combination thereof.

In one aspect, the size-randomized and shape-randomized nanopores ornanotubes form a vertically or parallel-aligned nanostructure array, ora combination thereof (see discussion, above).

The invention provides implants comprising a product of manufacture ofthe invention, including any of the nanostructures (e.g., nanotubes,nanopore-comprising structures) described herein.

The invention provides bioreactor comprising a product of manufacture ofthe invention, including any of the nanostructures (e.g., nanotubes,nanopore-comprising structures) described herein.

The invention provides artificial organs comprising a product ofmanufacture of any of claims 1 to 12.

The invention provides disease or toxin detection devices comprising aproduct of manufacture of the invention, including any of thenanostructures (e.g., nanotubes, nanopore-comprising structures)described herein. In one aspect, the disease or toxin detected is SARS,influenza (e.g., the so-called “bird flu”) or anthrax.

The invention provides orthopaedic or dental prostheses comprising aproduct of manufacture of the invention, including any of thenanostructures (e.g., nanotubes, nanopore-comprising structures)described herein.

The invention provides methods of making a product of manufacturecomprising a size-randomized and shape-randomized nanopore- ornanotube-comprising surface, the method comprising the following steps:(a) providing a composition comprising a Ti or Ti oxide surface; (b)depositing a semi-wettable coating on the Ti or Ti oxide surface byemploying lithographic patterning or by a thin film depositiontechnique, wherein the coating decomposes into nano- or micro-islands oflocal etch masks; and (c) chemical etching or electrochemicalanodization of the etch-masked surface, thereby generating asize-randomized and shape-randomized nanopore- or nanotube-comprisingsurface.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

The invention provides nano-scaled materials that exhibit extraordinaryphysical, mechanical and biological properties, which cannot be achievedby micro-scaled or bulk counterparts. The Ti and Ti alloys used in thecompositions of this invention are corrosion resistant, light, yetsufficiently strong for load-bearing, and are machinable. Because Ti andTi alloys are one of the few biocompatible metals which osseo-integrate,the compositions of the invention provide a template for direct chemicalor physical bonding with adjacent bone surface without forming a fibroustissue interface layer; e.g., in the orthopedic and dental implants ofthe invention; incorporating devices as described, e.g., in Handbook ofbiomaterial properties, ed. J. Black and G. Hasting, London: Chapman &Hall, 1998; B. D. Ratner et al., Biomaterials Science, San Diego,Calif.: Academic press; 1996.

The invention provides compositions allowing accelerated bone growth,which in some aspects is beneficial for fast recovery of patients withimplant operations for repair of joints, broken bones, or dentalimplants. In addition, accelerated cell growth using structured andconfigured biomaterials of this invention can be useful for a variety ofbio-applications. Biocompatible nanostructures of the invention can bemade to serve multi-functional roles to additionally accelerate bone andcell growth, its practical usefulness can be further enhanced.

The invention provides compositions and methods for cell growth onTiO.sub.2 nanotubule array structures as described herein; thecompositions of the invention can provide enhanced and designed patternsof cell and bone growth obtainable by modifying the Ti or TiO.sub.2 innanopore, nano-reservoir or nanotube configurations. For example, eventhe same type of cells have substantially varying cell size and shapefrom one cell to another, and some type of cells, especially the livercells, seem to grow better when co-cultured with other types of cells,such as endothelial or fibroblast cells which have significantlydifferent cell sizes. The invention provides compositions comprisingnanopore, nano-reservoir or nanotube configurations appropriate for thegrowth, differentiation and maintenance of a varied cell population,e.g., to form a functional tissue or organ, e.g., as in an artificialorgan, such as an artificial liver or kidney.

In this invention, improved biomaterials with nanostructures withsize-randomized (and also shape-randomized) nanopore or nanotubestructure are provided, which are especially suitable for co-culture ofhepatocytes with other cells. These biomaterials are made of Ti orTiO.sub.2 or equivalent structures, or made of other materials and thencoated with a biocompatible Ti or TiO.sub.2 film.

In one aspect, the size-randomized nanopore or nanotube structures ofthe invention have a sufficient distribution of pore entrance diameter(or average diameter if non-circular) to allow enhanced cell adhesionand accelerated growth of, for example by at least about 100/o, 20%,25%, 50%, 60%, 75%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or500% faster.

In one aspect, the desired distribution of the nanopore sizes in thenanostructure is such that at least one third of the pores have theiraverage diameter which is equal to or less than 10%, 20%, 25%, 50%6,60%, 70% or 75%, or equal to or less than 50% of the overall averagepore diameter, while another one third of the pores have their averagediameter which is at least 125%, 150%, 175%, 200%, 250%, 300%, 400% or500% the overall average pore diameter. As in some aspects, thesize-randomized nanopores or nanotubes have larger diameters, these willbe more suitable for efficient insertion and storage of biologicalagents in the nanopores.

The biological agents that can conveniently be stored in the nanoporesinclude growth factors, collagens, various proteins/biomolecules,nucleic acids (e.g., siRNA, DNA, RNA, vectors, genes), antibiotics,hormones, drugs such as cancer drugs or diabetes drugs, functionalparticles like magnetic, radioisotopes, dyes, metallic, ceramic, polymerparticles. The functional particles can be utilized for hyperthermia ormagnetic hyperthermia treatment of tumors, with the particles optionallyconjugated with other molecules for drug delivery, accelerated cell/bonegrowth, therapeutic treatments, etc.

Biomaterial structures of the invention can enable accelerated growth offunctional organ cells such as liver or kidney cells as well as otherstructural cells such as blood vessel cells, enzyme secretion vessels,periodontal, bone, teeth, or other hard tissue cells growth. Theavailability of such cultured cells can be useful for a variety ofapplications including i) organ-related therapeutic medical treatmentincluding liver or kidney disease treatment, ii) orthopedic, dental orperiodontal processes, iii) supply of cells for various research ortherapeutic purposes, and iv) disease diagnostic or toxicity testing ofnew drugs or chemicals.

The nanotube or nanopore structures of the invention can substantiallyimprove cell adhesion and growth kinetics; including adhesion ofanchorage-dependent cells such as osteoblasts, which is a crucialprerequisite to subsequent cell functions such as synthesis ofextracellular matrix proteins and formation of mineral deposits. In oneaspect, nanotube or nanopore structures of the invention comprisevertically or parallel-aligned nanostructure arrays; and they can havestructural advantages for reduced interfacial failure. In one aspect, avertically or parallel-aligned nanopore or nanotube array is fabricatedto be thin, e.g., less than 3000 nm, 2000 nm, 1500 nm, 1000 nm, 500 nm,400 nm, 300 nm, 200 nm, or less than 100 nm. In one aspect, nanopore ornanotube arrays of the invention can be made of the same material as thebase substrate material to ensure for strong bonding and mechanicalstability of the nanotube array, for example, TiO.sub.2-coverednanopores or nanotubes have a common element Ti shared with the implantsubstrate material titanium, thus providing a strong chemical bonding.

Referring to the drawings, FIG. 46A-46E schematically illustratesexemplary configurations of a size-randomized nanopore structure ofbiocompatible materials such as Ti or TiO.sub.2, with the size and shapeof the entrance of the pores having a distribution of dimensions asshown in FIGS. 46C-E, as compared to the uniform sized nanopores FIGS.46A and 46B. Such a distribution in size and shape of the nanopores ornanotubes can help achieving enhanced adhesion and accelerated growth ofvarious size and shape cells simultaneously, as illustrated in FIG. 46E.Such size-randomized nanopore or nanotube biomaterial (FIGS. 46C-E) canbe fabricated, according to the invention, by using various mask-guidedchemical etching fabrication or thin film deposition approaches.

In one aspect, the pre-patterned, e.g., random patterned, masks (togenerate pre-patterned, e.g., random patterned, pores or nanoreservoirs)are prepared by lithographic patterning of craters, e.g., byphotolithography (see. e.g., U.S. Pat. Nos. 6,030,266; 5,759,744;6,946,390), UV or deep UV and extreme UV lithography (see, e.g., U.S.Pat. No. 7,014,961), laser interference lithography (see, e.g., U.S.Pat. No. 5,726,524), e-beam lithography (see, e.g., U.S. Pat. No.6,956,333), imprint lithography (see, e.g., U.S. Pat. Nos. 7,027,156;6,842,229), particle multibeam lithography (see, e.g., U.S. Pat. No.6,989,546) or ion beam lithography (see, e.g., U.S. Pat. Nos. 6,949,756;6,414,307; 6,303,932 6,924,493), nano imprint lithography, and variousother known techniques, or by coated-layer decomposition approach aswill be described later in this specification. The random patternedmasks also can be prepared by techniques such as sol-gel method (see,e.g., U.S. Pat. No. 7,014,961): photolithographic patterning of aphotoresist layer by pattern-wise exposure to short-wavelengthultraviolet light through a pattern-bearing photomass, as described inU.S. Pat. No. 6,593,034; electrophoretic deposition and anodization.See, e.g., Lakshmi, et al. (1997) Chemistry of Materials, 9:2544-2550;Miao, et al. (2002) Nano Letters 2:717-720; Gong. et al. (2001) J.Materials Res. 16:3331-3334; Macak (2005) Chem. Int. Ed., 44:7463-7465.

Once the pre-patterned (including “random patterned”) craters are formedwith a desirable size, location and distribution, the implant orbio-substrate can be subjected to the chemical, electrochemical or ionetching to form desired size-randomized nanotube, nanopore ornano-reservoir structures, e.g., with aligned nanopores, nano-reservoirsor nanotubes.

Titanium nanotubes, nano-reservoirs or nanopores of the invention alsocan be formed by electrolytic anodization, for example using 5%hydrofluoric acid and applying approximately 10 to 20 volts ofpotential, and allowing several minutes to a few hours depending on thetemperature and other electrochemical process parameters. It is knownthat the resultant TiO.sub.2 nanotube diameter is dependent on theanodization voltage. The dimension of approximately 100 to 150 nm isoften not large enough to incorporate biological agents andbiomolecules, and is also not large enough for a substantial portion ofcells (typically many micrometers in size) to go into the pore and forma locked-in structure, although a portion of the filopodia branches canget into the pore and improve cell adhesion and cell growth kinetics;thus, in one aspect, the structures of the invention include pores ortubes larger than 100 to 150 nm. From this point of view, in one aspect,the size-randomized nanopore or nanotube structures of the inventionoffer an advantage in that a substantial portion of the pores can bemade larger than approximately 150 nm, as many of the processes of theinvention, including lithographically defined masks, semi-wettableballed-up island masks or nanoparticle masks, mask-island-guided ormask-hole-guided etching or anodization, can generate compositionshaving all or a portion of their nanostructures (e.g., nanopores,nano-reservoirs or nanotubes) larger than 150 nm. In alternativeaspects, larger-diameter-containing nano-reservoir-, nanotube- ornanopore-comprising structures have diameters greater than approximately150 nm, 175 nm, 200 nm, 250 nm or 300 nm or more, in at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45% or 50% more of the total surface areafraction, or up to at least 90% or more of the total surface areafraction.

Exemplary material that can be processed into desired size-randomized orsize-designed nano-reservoir-, nanotube- or nanopore-comprisingstructures include alloys containing Ti with at least 10%, 15%, 20%,25%, 30%, 35%, 40%, 45% or 50% more weight %. TiO.sub.2 can also beutilized. Other related materials such as Zr, Hf. Nb, Ta, Mo, W, andtheir oxides, or alloys of these metals and oxides by at least about10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% o or more weight % can beused. Other materials such as Si, Si oxide, carbon, diamond, noblemetals (such as Au, Ag, Pt and their alloys), polymer or plasticmaterials, or composite metals, ceramics or polymers can also beutilized to produce and use similar desired surface configurations,e.g., for the bio implant and cell growth applications of thisinvention; where in one aspect, these materials are used as long as acoating of Ti and Ti oxide, Zr, Hf. Nb, Ta, Mo, W and their oxides, aswell as their alloys, with a thickness of at least 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 or more nm and the coating coverage of at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70% or 80% more of the totalsurfaces is provided.

Shown in FIG. 47A is an exemplary method of fabricating size-randomizednanopores or nanotubes on non-flat surface such as Ti wire, mesh, orfoam, or on a flat surface such as on a Ti plate or sheet, by usingsemi-wettable or island-forming coating as a local mask. The coatingillustrated in FIG. 47B can be either a polymer-based material, metal oralloy based material, or a salt based material. A thin coating ofpolymer can be deposited by a number of well known techniques such asdip coating, spray coating, brush coating, evaporation, chemical vapordeposition, or spin coating (if the object has flat surfaces). Thepolymer coated object can be either dried to remove the solvent or wateror heated to alter the wetting characteristics in such a way that thecontinuous coating is broken up into islands, as illustrated in FIG.47C. The metal or alloy based material can be deposited by sputtering,electroplating, evaporation, chemical vapor deposition, or plasma spray.For a non-flat object, rotation of the object during physical vapordeposition id desired to make sure that most of the surfaces are coveredby the deposited material. The metal coated object is then heated to arelatively high temperature of e.g., 300-900 degree. C. to make themetal ball up into islands to reduce the overall free energy. The metalor alloy coating material should be selected in such a way that thedegree of alloying with the base metal is minimal so that extensivewetting does not occur. The salt based material can be either a aqueoussolution or solvent solution, optionally mixed with viscosity enhancersuch as polyvinyl alcohol, can be applied by dip coating or spraycoating, brush painting, and heated to form island masks. The salt basedcoating material can also be applied by evaporation or sputtering,followed by heating to decompose the salt and form mask islands.

The desired thickness of the coating applied depends on the desired sizeof the islands (masks) since a thinner coating results in smallerdiameter islands. The degree of wettability of the coating material onthe object also determines the resultant size of the islands. Thedesired thickness of the coating is typically in the range of anywherebetween about 2-2000 nm, or about 5-200 nm.

FIG. 48A-48E schematically illustrates an alternative exemplary methodof size-randomized nanopore or nanotube formation using non-flat orflat, TiO.sub.2 covered surface such as a Ti wire, mesh, foam, plate, orsheet, by using semi-wettable or island-forming coating as a local mask.Here a pre-TiO.sub.2-covered (oxidized or anodized) Ti material, FIG.48A, is utilized as the base material, and processed similarly as in thecase of FIG. 47. However, this exemplary chemical or electrochemicaletching is carried out through the TiO.sub.2 except the island-coveredregions to form craters, which is then followed by chemical etching oranodization to form size-randomized nanopores or nanotubes asillustrated in FIG. 48B-D.

Alternatively, a polymer-containing or an aqueous solution containingnano particles of coating material (polymer, metal or salt) can beapplied on the object by dip coating or spray coating, and then heatedto decompose and form islands of masks as illustrated in FIG. 49A-C.Either chemical etching or anodization through mask islands, orformation of Ti-oxide coating except the mask islands followed bychemical etching or anodization can be utilized to obtain the desirednanopore or nanotube array structure as illustrated in FIG. 49D.

Instead of Ti and Ti-alloy based wire, mesh, foam, plate or sheet, theuse of other related materials such as Zr, Hf. Nb, Ta, Mo, W, and theiroxides, or alloys of these metals and oxides by at least 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 60%, 70% or 80% more weight % is notprohibited for the size-randomized nanopore or nanotube structures inFIGS. 46 to 49. Other materials such as Si, Si oxide, carbon, diamond,noble metals (such as Au, Ag, Pt and their alloys), polymer or plasticmaterials, or composite metals, ceramics or polymers can also beutilized to produce and use similar desired surface configurations forbio implant and cell growth applications; where in one aspect, thesematerial are used as long as a coating of Ti and Ti oxide, Zr, Hf; Nb,Ta, Mo, W and their oxides, as well as their alloys, with a thickness ofat least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nm and the coatingcoverage of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%,70% or 80% more of the total surfaces is provided.

FIG. 50A-50F represents a randomized pore size and shape fabricated bythin film deposition. A proper selection of sputtering pressure andtemperature can introduce porous or rough microstructure in depositedthin films. The self-shadowing effect of the obliquely deposited thinfilm material, e.g., by evaporation can be used to form highly porous orrough films. The sputtering deposition can be carried out by DC, pulseDC, RF sputtering, or ion beam deposition methods. The evaporation canbe done by thermal or electron beam evaporation process. Depending onthe deposition conditions, a smooth continuous film, rough topologyfilm, or highly porous structure can be obtained, e.g., as described byThornton (1986) J. Vac. Sci. Technol. A4(6):3059; Meng et al“Investigations of titanium oxide films deposited by dc active magnetronsputtering in different sputtering pressures”, Thin Solid Films 226, 22(1993), by K. Robbie et al “Fabrication of thin films with highly porousmicrostructure”, J. Vacuum Science & Technology, 13(3), 1032 (1995):Rodriguez et al, “Reactively sputter deposited titanium oxide coatingswith parallel Penniform microstructure”, Adv. Mater. 12(5),341 (2000).

Exemplary highly porous structures of the invention obtained by thinfilm deposition are schematically illustrated in FIG. 50A-50F. A porousTi thin film can be deposited and oxidized or a porous TiO.sub.2 filmcan directly be deposited as shown in FIGS. 50A-C as a size-randomizednanoporous biomaterial for enhanced cell adhesion and bone growth.Alternatively, a rough or faceted surface topology can be provided byadjusting thin film deposition process parameters, which is thensubjected to anodization to form a vertically porous size-randomizednanopore or nanotube structure as illustrated in FIGS. 50D-(f). In bioapplications, these structures can be modified by inserting/storingbiological agents in the size-randomized nanopores as shown in FIG.51A-51F.

The biological agents that can conveniently be stored in these nanoporesinclude growth factors, collagens, various proteins/biomolecules,nucleic acids (e.g., genes, DNAs, RNA, siRNAs, vectors) antibiotics,hormones, drugs such as cancer drugs or diabetes drugs, functionalparticles like magnetic, metallic, ceramic, polymer particles,radioisotopes, and the like. In one aspect, the functional particles areutilized for hyperthermia or magnetic hyperthermia treatment of tumors,with the particles optionally conjugated with other molecules for drugdelivery, accelerated cell/bone growth, therapeutic treatments, etc.

In FIG. 52A-52C, the size-randomized porous TiO.sub.2 structure isdirectly formed by reactive sputtering process or by evaporation in thepresence of oxidizing atmosphere such as by supplying some partialpressure of oxygen during film deposition. These size-randomized porousTiO.sub.2 structures can be altered into improved bio materials byinserting/storing biological agents similarly as in the case of FIG.51A-51F for enhanced cell adhesion and bone growth.

Referring to FIG. 53A-53D, the figure schematically illustrates thelithographically created random-sized pores or nanotubes according tothe invention. FIGS. 53A and 53B as-fabricated, 53C biological agentinserted into the nanopores, 53D functional nanoparticles added into thenanopores.

The size-randomized and pattern-randomized masks can be prepared bylithographic patterning of photoresist or e-beam resist layer such asPMMA (polymethylmecarthrylate) or any other positive or negativepatternable polymer, as illustrated in FIGS. 46C-E and FIGS. 53A and B.Lithography techniques such as photolithography (including UV, deep UVand extreme UV lithography, laser speckle interference lithography,etc.), e-beam or ion beam lithography, nano imprint lithography, andvarious other known techniques can also be utilized. The patternedgeometry can be circular, oval or any irregular shape.

Cells of various sizes and shapes, especially in the case ofco-culturing for liver cell growth, are cultured with enhanced celladhesion, faster proliferation kinetics, and in healthier conditions dueto the size-randomized and shape-randomized nanopore or nanotubestructure on the surface of the bio implants or bio substrates of theinvention. In one aspect, size-randomized biomaterials also allowrelatively easier insertion/storage of biological agents such as growthfactors, collagens, various proteinsibiomolecules, genes, DNAs,antibiotics, hormones, or drugs, as well as functional particles such asmagnetic nanoparticles, photoluminescent particles, and other metallic,ceramic, polymer particles. This is due to the presence of some largerdiameter nanopores or nanotubes in the distribution of the random poresizes in the biomaterials. These characteristics are schematicallyillustrated in FIGS. 53B-D.

In one aspect, size-randomized nanopore or nanotube structuresincorporate re-entrant, gradient or corrugated pore wall configurations,as illustrated schematically in FIG. 31A-31D and FIG. 34.Size-randomized nanopore or nanotube structures with at least 50% of thepores having a re-entrant or lock-in nanostructure can also be utilized,as illustrated in the figure. The re-entrant characteristics of thenanopore or nanotube entrance is arbitrarily defined here as having anaverage entrance diameter (or average pore size if they are notcircular) by at least about 10%, 15%, 2%, 25%, 30%, 35%, 40%, 45%, 50%,60%, 70% or 80% smaller than the rest of the nanopore or nanotubedimension so that the cells or bones grown are mechanically more firmlyattached. FIG. 54A-54D schematically illustrates size-randomizednanopores or nanotubes with re-entrant, gradient or corrugated pore wallconfiguration, together with biological agents stored in the nanopores.

In one aspect, size-randomized nanopore or nanotube structures of theinvention have dual structured biomaterials comprising a micro or macropores in combination with surfaces covered with randomized andfiner-scale TiO.sub.2 nanotubes, as illustrated in FIG. 35A-35C. In oneaspect, the dual-sized structure allows micro- or macro-scale growth ofbones essentially completely fill the large pores to guard against theslippage or mechanical failure of grown bones against tensile or shearstresses, while enabling accelerated osteoblast cell growth on thenanotube-covered surface of the implants. In one aspect, dual structurescomprise any combination of nano-, micro- or macro-pores, or, dualstructures comprise a re-entrant configuration having an averagediameter (or equivalent diameter if the pores are not circular) in therange of between about 0.5-1,000.mu.m, or about 1-100.mu.m; and, in oneaspect, dual structures are constructed as a nanostructure comprisingsize-randomized nanopores or nanotubes having an average pore diameterin the range of between about 30-600 nm. In one aspect, the relativeratio of the micro/macro pores vs nanopores in the dual structure issuch that the micro/macro pores occupy at least 10%, 11%, 12%, 13%, 14%,15%, 20% o, 25%, 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75% or more area fraction of the implant orbio-substrate surface; and in one aspect, between about at least 20% butless than 50% to maximize the accelerated cell culture or bone growthvia the nanopore portion of the surface.

FIG. 40A-40E illustrates a size-randomized nanopore or nanotube array onvarious surfaces of the invention by guided etching using variousexemplary techniques, e.g., as described in relation to FIGS. 46 to 54,FIG. 31A-31D and FIG. 35A-35C. The size-randomized nanopore or nanotubearray can be formed on various exemplary surfaces i.e., on a flatsurface (FIG. 40A), on a coarse-patterned surface (FIG. 40B), onparallel Ti sheet or wire array (FIG. 40C) which can be useful forthree-dimensional cell or organ structure, on wire mesh, wire bundle orscaffold type highly porous metal foam (e.g., made of Ti or its alloy)(FIG. 40D), and on re-entrant cavity surface (e.g., pre-shaped Ti orTi-coated Si) (FIG. 40E).

The structures illustrated in FIGS. 40C and D are particularly desirablefor accelerated co-culturing of healthy three dimensional cells andorgans such as desired for liver cell growth. These 3-D cultured cellscan be prepared using parallel Ti sheet, wire array or mesh and foamarrays or the invention with size-randomized nanopore or nanotube arraysurface structures. These structures can be useful for a variety ofapplications, including in vivo implanting of cells or organs. A patientcan be supplied with in vitro cultured and in vivo implanted,three-dimensional functional cells such as liver, kidney, or bloodvessel cells or organs. Three-dimensionally cultured bones or tissuescan also be utilized as dental, periodontal or orthopedic body implants.

To obtain relatively large surface area for cell growth, the desiredthickness of the titanium metal ribbon, or the desired diameter of themetal wire or foam in FIGS. 40C and D is in the range of about10.mu.m-50,000.mu.m, or about 25-2,500.mu.m. The desired volume fractionof the metal for the given targeted cell volume at the end of theplanned cell culture period is at least about 10%, 11%, 12%, 13%, 14%,15%, 20%, 25%, 30% or more in volume, or at least about 30%, 40% or 50%in volume.

In one aspect, each of the ribbon surface is made to contain an alignednanopore or nanotube array with a preferred diameter being in the rangeof 10-1000 nm, or about 30-300 nm, or about 60-200 nm in diameter; andin one aspect, a desired height being in the range of about 40-2000 nm,or about 100-400 nm; and in one aspect, a desired angle is a verticalwith an allowance of about a 10, 20, 25, 30 or 35 degree variation offthe perpendicular axis.

In one aspect, the shape of these nanostructure-surface ribbons isdesirably straight so that the metal arrays can be optionally pulled outafter a desired volume of the cells are cultured. In one aspect, thecultured growth will continue after the metal wire or ribbon arraytemplate structure is pulled out, and any minor surface damage or thethin empty gap created by the vacated template will be repaired/filledby growing cells. Such a three-dimensionally cultured cell volume in anaccelerated manner can be useful for a variety of applications includingcreation of partial or full artificial organs of e.g., liver, kidney,bone, periodontal tissue, blood vessel cells, skin cells, stem cells,and other human or animal organ cells etc.

In one aspect, the 3-D cultured cells are in any orientation, i.e.,horizontal, vertical or upside down depending on specific needsespecially in the in vivo culture environment, for example, in the caseof organ implants in human, animal, or xenotransplantation of humanorgans which are temporarily cultured in animals prior to humanimplantation. FIG. 55A-55E schematically illustrates the enhanced growthof various sized cells and three-dimensional organs on thesesize-randomized biomaterials.

In one aspect, various biological agents or functional nanoparticles areadditionally be stored in the surface nanopores, nanoreservoirs ornanotubes on these various shaped bio-substrates or bioimplants shown,e.g., in FIG. 40A-40E and FIG. 55A-55E. The functional nanoparticlesinclude magnetic particles, novel metal or SPR (surface plasmonresonance) based photoluminescent particles, quantum dots, fluorescenceparticles, bio-conjugated particles, which are added for the purpose ofaccelerated cell/bone growth, protein harvest (as a result ofaccelerated cell growth, proliferation, and secretion), delivery ofdrugs, genes, chemicals, therapeutics, etc.

FIG. 56A-56C illustrates exemplary processes of guided etch nanopatterning of size-randomized nanopores or nanotubes on Ti and relatedbioimplant or bio-substrates. Random size patterning techniques onnon-flat surfaces using conformable or stretchable elastomeric masksheet (FIG. 56A), using elastomeric roll stamping (FIG. 56B), andelastomeric flat stamping on large area surfaces (FIG. 56C) aredescribed. In one aspect, the invention provide for use of nanoimprinting to pre-pattern craters on Ti or TiO.sub.2 surface for guidedsynthesis of size-randomized or shape-randomized TiO.sub.2 nanotubes andnanopores. Such nano imprint lithography technique is convenient formass production of a large number of implants or bio substrates assimple stamping operation can accomplish the pre-patterning of craterswithout resorting to complicated and often costly photolithography ore-beam lithography patterning of each part. In one aspect, an advantageis the ability to use elastomeric stamp with compliance, which allowsreliable stamping on large-area, nominally-flat surfaces of Ti or otherbiocompatible substrates and implants in which the flatness is notalways guaranteed to ensure reliable stamping over a large sample area.As bio implant parts are more often than not in a non-flat surfacegeometry, the invention provides a convenient and effective means offabricating the pre-patterned craters for guided introduction ofsize-randomized nanopores or nanotubes on the surface of random shapedTi implants. FIG. 56A illustrates exemplary processes of guided etchnano patterning of size-randomized patterns on non-flat surfaces using aconformable or stretchable elastomeric mask sheet, while FIG. 56Billustrates an exemplary use of elastomeric roll stamping on round rodconfigured implants. FIG. 56C illustrates the exemplary nanoimplantprocess of size-randomized patterns on flat surfaces of implants orbio-substrates.

FIG. 11A-11C schematically illustrates various exemplary in vivo or exvivo applications of the exemplary size-randomized pored biomaterialscapable of accelerated cell/bone growth or functional drug delivery andtherapeutics. Various exemplary nanopore or nanotube structures of theinvention, e.g., as described in relation to FIGS. 46 to 54, FIG.31A-31D. FIG. 35A-35C, FIG. 40A-40E, can be utilized for thesebiomedical device applications. Examples shown include orthopaedic anddental implants, cell or organ implants, drug delivery devices such ascontrolled release of insulin by magnetic actuation, artificial liverdevices, drug-protected stents (e.g., to prevent/minimize restenosis) orother tubules inserted into blood vessels and in various other bodyparts, and therapeutic devices such as magnetic field induced localheating for cancer treatment. Cell growth inhibiting drugs can also beinserted in the nanopores or nanotubes of implants (e.g., drug deliverymodules) to prevent/minimize scar tissue formation.

Illustrated in FIG. 15 is an exemplary magnetically actuated, on-offcontrollable or programmable, remote drug release device comprising theexemplary biomaterials. Remote magnetic field such as approximately 100KHz AC magnetic field can be utilized to activate the drug release bypreferential heating of the magnetic nanoparticles stored in thenanopores of the exemplary implants, thus allowingtemperature-gradient-induced drug movement from the nanopores into thehuman body. Suitable magnetic nanoparticles for such use includebiocompatible iron-oxide particles of magnetite (Fe.sub.3O.sub.4) ormaghemite (.gamma.-Fe.sub.2O.sub.3) in the particle size regime of 5-50nm in average diameter.

In one aspect, a mechanical agitation and induced movement of themagnetic particles themselves in the pores containing the stored drugscan be utilized as an alternative mechanism of drug release. In thiscase, a rather lower frequency AC magnetic field actuation with agradient magnetic field is preferred in order to allow movement ofmagnetic particles against the viscosity of the fluid in the nanopores.Exemplary frequency ranges are approximately 1 to 10,000 Hz, or about 10to 1,000, or about 10 to 100 Hz.

In these aspects, the drugs are released any time of the day only whendesired, and can be turned off completely when it is no longer needed,so the side effects of drugs are minimized. A similar magnetic particleand external field combination can also be utilized for cancertreatment, for example, through an implant devices comprising theexemplary nanoporous structure with size-randomized nanopores ornanotubes placed in the vicinity of cancerous regions, for example, inthe liver containing inoperable distribution of recurring/growing cancercells. A repeated hyperthemia treatments. e.g., over many months, byon-off type continual activation of external magnetic field cangradually kill the cancer cells near the cancerous regions.

FIG. 57A-57B illustrates an exemplary method of harvesting cellscultured in an accelerated way using the exemplary biomaterialscontaining size-randomized nanopores or nanotubes. The exemplarynanopore or nanotube structures comprising TiO.sub.2 or relatedmaterials accelerate growth of certain types of cells. The increasednumber of cells generated by such a device can be useful for acceleratedsupply of cells, especially rare cells such as stem cells for variousR&D or therapeutic uses. As illustrated in FIG. 57A, the cells arecultured in a biocompatible environment with needed nutrient media. Thecells so proliferated on the exemplary nanopore or nanotube arrays arethen harvested and supplied for other uses. In addition to the cellsthemselves, it is sometimes desirable to collect secretions from thecells for various useful R&D or therapeutic applications, for example,increased amount of proteins generated as a byproduct of cellproliferation and functioning.

Cultured cells need to be taken off the culture substrates for otheruses. One exemplary method of harvesting the grown cells off thebiomaterial substrate is to use a process known as “trypsinization”.Once cells are grown completely on whole surface of cell culture flask,the media fluid is removed by suction. After rinsing of the cells twicewith PBS (phosphate buffer solution), trypsin is added to detach thecell from the surface. In general, approximately 2-3 ml of trypsin isused for detaching cells grown on 10 cm.sup.2 cell culture dish. After afew minutes, most of the cells are detached from the surface, asillustrated in FIG. 57B. After adding approximately 10 ml of new medium,this fluid containing the detached stem cells is poured into acentrifuge tube. After centrifugation at appropriate rotation speed andtime, all of the cells are separated. The medium is removed by suction,and approximately 1 ml of new media is added for storage of theharvested cells or for additional culture. To estimate the number ofproliferated cells, trypan blue assay are employed in conjunction withhematocytometry. The cells can also be in situ proliferated on theimplant surfaces as the TiO.sub.2 as well as Ti substrate arebiocompatible.

In one aspect, size-randomized Ti-oxide nanopore or nanotube structureis also useful for carrying out fast diagnosis and detection of certaintypes of cells such as diseased cells, or cells exposed to allergens,irritants, toxins, poisons or infectious agents, e.g., bacteria. An X-Ymatrix subdivided array of size-randomized nanopore or nanotube arraystructure can be produced as illustrated in FIG. 44. Any size-randomizednanopore or nanotube structures of the invention, e.g., as described inrelation to FIGS. 46 to 54, FIG. 31A-31D, FIG. 35A-35C, FIG. 40A-40E,can be utilized for accelerated cell detection/diagnosis. For diagnosisof diseases (e.g., epidemic diseases) or, detecting exposure tobiological or chemical warfare agents (e.g., bacteria or viruses), theinvention provides rapid detection devices, even when the availablequantity of the cells is relatively small. Each of the exemplarydetection elements in FIG. 44 contains a multiplicity of thesize-randomized nanopores or nanotubes on which various types of cellsto be analyzed are placed and allowed to rapidly proliferate to asufficient number for easy detection.

For analysis of cell types, various exemplary techniques, illustrated inFIG. 18A-18C can be used, including 18A optical detection of morphologyand size (using a microscope, fluorescent microscope, or CCD camerasensing of fluorescent or quantum dot tagged cells), 18B chemical orbiological detection (e.g., based on signature reactions), 18C magneticsensor detection (e.g., by using magnetically targeted antibody and itsconjugation with certain types of antigens).

An exemplary application of the invention's bio-chip apparatus, e.g., asillustrated in FIG. 44, utilizes a base structure to culture liver cellsfor testing of new drugs, as illustrated in FIG. 58, which illustratesan exemplary bio-chip device for toxicity testing of drugs or chemicals,with the device comprising an array of cultured liver cells grownrapidly and healthy manner on the biomaterials containingsize-randomized nanopores or nanotubes.

Similarly as in the cell detection/diagnosis applications, variousexemplary nanopore or nanotube structures of the invention, e.g., asdescribed in relation to FIGS. 46 to 54, FIG. 31A-31D, FIG. 35A-35C,FIG. 40A-40E, can be utilized for the liver cell culture and toxicitytest applications. A variation of the embodiments of FIG. 44 and FIG. 58include having a parallel wire, rod or plate shape base structure in avertically (or near vertically) arranged configuration (with each rod orplate base containing parallel, yet laterally spaced apart nanopore ornanotube array) in order to make the 3-D culture more effective forfurther increased speed of cell proliferation.

As these exemplary three-dimensionally structured, nanopore or nanotubearrays enable a rapid and healthy culture of liver cells in a desirablethree-dimensional configuration, the apparatus can be utilized for theimportant task of testing drug toxicity or chemical toxicity. Anexemplary bio-chip comprises an array of healthy, three-dimensionallycultured liver cells, e.g., 10.times.10, 100.times.100, or1000.times.1000 cells as sensing elements, can thus allow simultaneousevaluation of many drugs for much accelerated screening and developmentof biologically acceptable drugs. Likewise, many chemicals, polymers,injection fluids, and composites that may be useful for in vivoapplications can be rapidly tested for toxicity using the exemplarydevice of FIG. 58. The accelerated, healthy liver cell growth device ofFIG. 58 can also be utilized as the basis of artificial liver devicesfor patients waiting for transplant or as a temporary aid to liverfunction after transplant. The invention provides other functionalorgans applications, such as artificial kidneys.

It is understood that the above-described embodiments are illustrativeof only a few of the many possible specific embodiments which canrepresent applications of the invention. Numerous and varied otherarrangements can be made by those skilled in the art without departingfrom the spirit and scope of the invention. For example, the materialsinvolved do not have to be Ti oxide nanotubes on Ti-based metals, as thenanotubes and the substrate that the nanotubes are adhered to can beother biocompatible materials or non-biocompatible materials coated withbiocompatible and bioactive surface layer such as Ti, or coated withbiocompatible but bio-inert surface layer such as novel metal or polymerlayer. Also, a thin coating of biomolecules or chemical molecules canoptionally be applied on the surface of TiO.sub.2 nanopores or nanotubesto further enhance the attachment of cells.

Therefore, it should be understood that the invention can be practicedwith modification and alteration within the spirit and scope of theappended claims. The description is not intended to be exhaustive or tolimit the invention to the precise form disclosed. It should beunderstood that the invention can be practiced with modification andalteration and that the invention be limited only by the claims and theequivalents thereof.

VI. Summary

The invention provides products of manufacture, e.g., bio-assemblies, ordevices comprising cells, including mixed cell systems, that can be usedas artificial tissues or organs, as chemical testing systems, asproduction factories for biological agents or for any research ordevelopment purpose. For example, the compositions (e.g., devices,products of manufacture) and methods of the invention can be used fordetecting a toxin, a poison, an allergen, a biological warfare agent, aninfectious disease agent or an irritating agent. In one aspect, thedetecting or sensing device comprises providing an X-Y matrix subdividedarray of a plurality of nanotubes. The invention provides methods fordetecting the effect of a compound on a cell or a tissue or organcomprising contacting a detecting device of the invention with a testmaterial selected from the group consisting of a chemical, a toxin, apoison, a cosmetic, a food, a natural product, an allergen, an irritant,a biological warfare agent, a polymer and an injection fluid.

For example, the invention provides devices, products of manufacture andstructures (e.g., bio-assemblies, such as 2-D or 3-D cell-comprisingdevices as implants or as cell factories or biological agent productionsources) comprising titanium dioxide-comprising structures comprisingnanotubes or nanopores of approximately equal size, e.g., less thatabout 100 nm or comprising macroscopic interlocking structurescomprising “openings”, reservoirs or pores which themselves may comprisenanotubes or nanopores; or comprising “dual structures”, or comprising“interlocking structures” which themselves may comprise nanotubes ornanopores; or comprising biocompatible vertically aligned nanotube arraystructures, including accelerated cell growth structures; or comprisinga lock-in nanostructure comprising a plurality of nanopores ornanotubes, wherein the nanopore or nanotube entrance has a smallerdiameter or size than the rest (the interior) of the nanopore ornanotube (e.g., to exhibit a re-entrant configuration); or comprisingdual structured biomaterial comprising micro- and/or macro-pores andnanostructures (e.g., nanopores or nanotubes as described herein); ordevices and structures comprising random sized pores, e.g., includingpores of less than 100 nm; or, devices and structures comprising anycombination, or all, of these structures.

For example, a product of manufacture of the invention can furthercomprise a two or a three-dimensional array of the invention, or abiocompatible vertically aligned nanotube array structure of theinvention, or an array of the invention, or a lock-in nanostructure ofthe invention, or a dual structured biomaterial of the invention, or anycombination thereof, e.g., a combination of two or three or more or allof these structures/compositions of the invention.

An implant or artificial organ of the invention can comprise a productof manufacture of the invention, a two or a three-dimensional array ofthe invention, or a biocompatible vertically aligned nanotube arraystructure of the invention, or an array of the invention, or a lock-innanostructure of the invention, or a dual structured biomaterial of theinvention, or any combination thereof, e.g., a combination of two orthree or more or all of these structures/compositions of the invention.

A bioreactor of the invention can comprise a product of manufacture ofthe invention, a two or a three-dimensional array of the invention, or abiocompatible vertically aligned nanotube array structure of theinvention, or an array of the invention, or a lock-in nanostructure ofthe invention, or a dual structured biomaterial of the invention, or anycombination thereof, e.g., a combination of two or three or more or allof these structures/compositions of the invention.

An artificial tissue or organ of the invention can comprise a product ofmanufacture of the invention, a two or a three-dimensional array of theinvention, or a biocompatible vertically aligned nanotube arraystructure of the invention, or an array of the invention, or a lock-innanostructure of the invention, or a dual structured biomaterial of theinvention, or any combination thereof, e.g., a combination of two orthree or more or all of these structures/compositions of the invention.

A disease detection device of the invention can comprise a product ofmanufacture of the invention, a two or a three-dimensional array of theinvention, or a biocompatible vertically aligned nanotube arraystructure of the invention, or an array of the invention, or a lock-innanostructure of the invention, or a dual structured biomaterial of theinvention, or any combination thereof, e.g., a combination of two orthree or more or all of these structures/compositions of the invention;wherein optionally the disease detected is influenza (flu), SARS oranthrax.

An orthopedic or dental prosthesis of the invention can comprise aproduct of manufacture of the invention, a two or a three-dimensionalarray of the invention, or a biocompatible vertically aligned nanotubearray structure of the invention, or an array of the invention, or alock-in nanostructure of the invention, or a dual structured biomaterialof the invention, or any combination thereof, e.g., a combination of twoor three or more or all of these structures/compositions of theinvention.

For example, the invention provides bio-assemblies comprising culturedanimal or human cells, tissues and/or bones attached and grown (and insome aspects, rapidly grown) on biocompatible vertically alignednanotube array structures with laterally separated nanotube arrangementswherein (a) the cultured cells, tissues and bones can be liver cells,kidney cells, nerve cells, myocytes, odontoblasts, dentinoblasts,cementoblasts, enameloblasts, odontogenic ectomesenchymal tissue,osteoblasts, osteoclasts, fibroblasts, and other cells and tissuesinvolved in odontogenesis or bone formation, stem cells, supportive softtissues such as muscles, skin cells, tendons, fibrous tissues,periodontal tissues, fat, blood vessels and/or hard tissues such as boneand teeth, either as a single cell type culture or as a co-culture of atleast two or more types of cells together; (b) the biocompatiblevertically aligned nanotube array structure is strongly adhered on asubstrate (e.g., a metallic substrate), and in one aspect has an outerdiameter of nanotubes in the range of between about 10 to 1000 nm, or 30to 300 nm, or 60 to 200 nm; and in one aspect, the inside diameter isopen with a diameter of at least about 10%, 20%, 30%, 40% or 50% or moreof the outer diameter; and in one aspect the desired height of thetubules is in the range of between about 40 to 800 nm, or 100-400 nm;and in one aspect the aspect ratio is less than 10, 9, 8, 7, 6, 5, 4 or3; and in one aspect the desired range of variation in the verticalalignment angle within between about 0 to 45 degrees, or 0 to 30 degreesoff the vertical direction; and in one aspect, lateral spacing betweenadjacent nanotubes is in the range of between about 2 to 100 nm, or 5 to30 nm; and, (c) the biocompatible nanotube array is made of a materialcomprising: i) titanium oxide on Ti support, a titanium alloy oxide onthe alloy support which contains at least about 10%, 20%, 30%, 40% or50% or more weight % Ti; ii) non-titanium oxide nanotubes of Zr, Hf, Nb,Ta, Mo, W, or alloys of these metals, or any mixture of these amongthemselves or in an alloyed form with Ti; or iii) coated Si, Si oxide,carbon, diamond, noble metals (such as Au, Ag. Pt and their alloys),polymer or plastic materials, or composite metals, ceramics or polymers;and in one aspect, having a laterally separated nanotube configuration;and in one aspect, having a surface coating with Ti oxide, Ti alloyoxide, or an oxide of Zr, Hf, Nb, Ta, Mo, W and their alloys; and in oneaspect, comprising a coating thickness of at least 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 or more nm; and in one aspect the coating coverage of atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more of the totalsurfaces.

In one aspect, the cells maintained or grown in these devices of theinvention, e.g., the rapidly cultured cells, tissues and/or bones, haveaccelerated growth kinetics by at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% 100%, 200%, 300% or more, as compared to the plain Timetal surface without a nanotube or nanopore structure of the invention.

In one aspect, the aligned titanium oxide nanotubes adhered on titaniumhave a strong adhesion strength of anywhere between about 100 and 1000psi. e.g., at least 500, 600, 700, 800, 900 or 1000 or more psi.

In one aspect, devices of the invention further comprise nanostructurescomprising sodium titanate or titanium oxide fibers, which in one aspectare present at the tip of nanotubes, e.g., with a fiber thickness of atmost 10, 20, 30, 40 or 50 or more nm, e.g., for enhanced hydroxyapatiteformation in a living body (e.g., as an implant) or in a simulated bodyfluid.

In one aspect, devices of the invention comprise inside pores, which canbe located in the laterally separated nanotubes, and they can comprisebiologically active agents, e.g., therapeutic drugs, growth factors,proteins, enzymes, hormones, nucleic acids (e.g., RNA, DNA, genes,vectors), antibiotics or antibodies, or other substances (e.g., smallmolecules, radioisotopes, and the like). In one aspect, the inside poresof the laterally separated nanotubes comprise magnetic nanoparticles,metal and/or ceramic nanoparticles.

In one aspect, a nutrient fluid, in either in vivo human or animal bodyenvironments (e.g., as implants), or as in vitro cell cultureenvironments, is continuously supplied under the growing cells throughgap spacing between the laterally separated nanotubes; this can be inaddition to the general nutrient fluid supply route, e.g., the spacenear the top surface of the growing cells.

In one aspect, the device of the invention, e.g., the bioassembly, is apart of an orthopaedic implant, a dental implant, a periodontal implant,an organ (e.g., a liver) implant, a joint implant, a heart implant, andthe like. In one aspect, the device of the invention, e.g., thebioassembly, is a part of an artificial organ, e.g., an artificial liverdevice, an artificial kidney device and the like.

In one aspect, the inside pores of the vertically aligned nanotubes arereservoirs comprising one or more of various biologically active agents,e.g., therapeutic drugs, growth factors, proteins, enzymes, hormones,nucleic acids (e.g., RNA, DNA, genes, vectors), antibiotics orantibodies, or other substances (e.g., small molecules, radioisotopes,and the like), with the biologically active agents or other substancesslowly released from the nanotubes at a rate determined by the nanotubediameter and aspect ratio, and the viscosity and wetting characteristicsof the solution containing the biological agents or other substances.

In one aspect of the bio-assembly, the inside pores of the verticallyaligned nanotubes are reservoirs containing one or more of variousfunctional nanoparticles with a size of less than about 10, 20, 30, 40or 50 or more nm in average diameter, or in the range of between about10 to 300 nm, or 30 to 300 nm, or 60 to 200 nm. In one aspect,functional nanoparticles comprise magnetic, metallic, ceramic and/orpolymer particles.

In one aspect of the bio-assembly, both the biological agents or othersubstances (e.g., small molecules, radioisotopes, and the like) andfunctional nanoparticles are co-present. In one aspect of thebio-assembly, the structure of the nanotube ends is of partially cappedconfiguration to enhance retaining of the biological or functionalagents within the nanotubes and reduce the release rate until externallystimulated for accelerated release. The partial capping of the nanotubeends can be by oblique incident deposition of metallic, ceramic, polymeror other materials.

The invention provides controlled slow-release drug and functional agentdelivery devices comprising the structure of: (a) a biocompatiblevertically aligned nanotube array structure which is strongly adhered ona metallic substrate, and in one aspect, has an outer diameter ofnanotubes in the range of between about 10 to 1000 nm, or 30 to 300 nm,or 60 to 200 nm; and in one aspect the inside diameter is open with adiameter of at least about 10%, 20%, 30%, 40%, 50% or more of the outerdiameter; and in one aspect the desired height of the tubules is in therange of between about 40 to 800 inn, or 100 to 400 nm and in one aspectthe desired aspect ratio of less than 10, 9, 8, 7, 6, 5, 4, 3 or 2; andin one aspect the desired range of variation in the vertical alignmentangle within between about 0 to 45 degrees, or 0 to 30 degrees off thevertical direction; and in one aspect the desired lateral spacingbetween adjacent nanotubes in the range of between about 2 to 100 nm, or5 to 30 nm; and/or (b) a biocompatible nanotube array comprising: i)titanium oxide on Ti support, a titanium alloy oxide on the alloysupport which contains at least about 10%, 20%, 30%, 40% or 50% or moreweight % Ti; ii) non-titanium oxide nanotubes of Zr. Hf; Nb, Ta. Mo, W,or alloys of these metals, or any mixture of these among themselves orin an alloyed form with Ti; or iii) coated Si, Si oxide, carbon,diamond, noble metals (such as Au, Ag, Pt and their alloys), polymer orplastic materials, or composite metals, ceramics or polymers; and in oneaspect, having a laterally separated nanotube configuration; and in oneaspect, having a surface coating with Ti oxide, Ti alloy oxide, or anoxide of Zr, Hf, Nb, Ta, Mo, W and their alloys; and in one aspect,comprising a coating thickness of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 or more nm; and in one aspect the coating coverage of at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more of the total surfaces;and in one aspect the inside pores of the vertically aligned nanotubesare (or act as) reservoirs containing therapeutic drugs or otherchemicals; and in one aspect the therapeutic drugs or otherchemicals/substances are slowly released from the nanotubes at a ratedetermined by the nanotube diameter and aspect ratio, and the viscosityand wetting characteristics of the chemical/substance-containing (e.g.,drug-containing) solution stored within the nanotubes.

In one aspect, the drugs stored in the nanotubes are cancer treatmentdrugs, diabetes drugs, bone-growth accelerating drugs,tissue-formation-preventing drugs or anti-stenosis drugs.

In one aspect, the inside pores of the vertically aligned nanotubesadditionally contain one or more of various functional nanoparticleswith a size of less than 10, 20, 30, 40 or 50 or more nm in averagediameter, and can comprise magnetic, metallic, ceramic, or polymerparticles. In one aspect, the structure of the nanotube ends is ofpartially capped configuration to enhance retaining the biological orfunctional agents within the nanotubes. In one aspect, the partialcapping of the nanotube ends is by oblique incident deposition ofmetallic, ceramic or polymer materials.

The invention provides externally controllable drug delivery devicescomprising any structure of the invention, e.g., the nanotube ornanopore configurations described herein, and further comprisingcompositions to control the timing as well as the quantity of therelease of drugs or biological agents, e.g., compositions to control theinitiation, starting and stopping of the release by, e.g., ultrasonic ormagnetic agitation of the colloidal liquid containing the mixture of thedrug solution and the nanoparticles.

In one aspect, the nanoparticles are biocompatible magneticnanoparticles with an average diameter in the range of between about 5to 50 nm. In one aspect, the magnetic particles are magnetite(Fe.sub.3O.sub.4) or maghemite (.gamma.-Fe.sub.2O.sub.3). In one aspect,the onset and quantity of drug release is controlled by externalstimulation of the magnetic nanoparticles by alternating current (AC)magnetic field to induce agitation/movement of the magnetic particles orby heating of the drug-particle mixture due to the AC magnetic field. Inone aspect, the device is for cancer treatment using a combination ofchemotherapy and magnetic hyperthermia.

The invention provides an in vitro or ex vivo cell proliferation devicecomprising a culture medium and any structure of the invention (e.g.,comprising the nanotube or nanopore configurations described herein),wherein the cells are rapidly multiplied and harvested for supply forresearch, therapeutic, screening, biodefense or implant applications. Inone aspect, these cells are “functional cells,” such as liver cells,kidney cells, nerve cells, myocytes, adult stem cells, embryonic stemcells, supportive soft tissues such as muscles, tendons, fibroustissues, periodontal tissues, fat, blood vessels, and hard tissues suchas bone and teeth. In one aspect, cultured cells are harvested bytrypsinization. In one aspect, the proliferation of the cells isaccelerated by the controlled or externally initiated release ofbiological or functional agents, as described herein.

The invention provides analytical cell diagnostic biochip systemscomprising a cell culture medium, the intended cells to be analyzed, theaccelerated cell growth substrate, and a cell growth monitoringdetection device wherein the cell growth substrate comprises (a) abiocompatible vertically aligned nanotube array structure of theinvention, e.g., one which is strongly adhered on a metallic substrate;and in one aspect and in one aspect, has an outer diameter of nanotubesin the range of between about 10 to 1000 nm, or 30 to 300 nm, or 60 to200 nm; and in one aspect the inside diameter is open with a diameter ofat least about 10%, 20%, 30%, 40%, 50% or more of the outer diameter;and in one aspect the desired height of the tubules is in the range ofbetween about 40 to 800 nm, or 100 to 400 nm; and in one aspect thedesired aspect ratio of less than 10, 9, 8, 7, 6, 5, 4, 3 or 2; and inone aspect the desired range of variation in the vertical alignmentangle within between about 0 to 45 degrees, or 0 to 30 degrees off thevertical direction; and in one aspect the desired lateral spacingbetween adjacent nanotubes in the range of between about 2 to 100 nm, or5 to 30 nm; and/or (b) a biocompatible nanotube array comprising: i)titanium oxide on Ti support, a titanium alloy oxide on the alloysupport which contains at least about 10%, 20%, 30%, 40% or 50% or moreweight % Ti; ii) non-titanium oxide nanotubes of Zr, Hf, Nb, Ta. Mo, W,or alloys of these metals, or any mixture of these among themselves orin an alloyed form with Ti; or iii) coated Si, Si oxide, carbon,diamond, noble metals (such as Au, Ag, Pt and their alloys), polymer orplastic materials, or composite metals, ceramics or polymers; and in oneaspect, having a laterally separated nanotube configuration; and in oneaspect, having a surface coating with Ti oxide, Ti alloy oxide, or anoxide of Zr, Hf, Nb, Ta, Mo, W and their alloys; and in one aspect,comprising a coating thickness of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 or more nm; and in one aspect the coating coverage of at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more of the total surfaces;and in one aspect the inside pores of the vertically aligned nanotubesare (or act as) reservoirs containing therapeutic drugs or otherchemicals; and in one aspect the therapeutic drugs or otherchemicals/substances are slowly released from the nanotubes at a ratedetermined by the nanotube diameter and aspect ratio, and the viscosityand wetting characteristics of the chemical/substance-containing (e.g.,drug-containing) solution stored within the nanotubes. In one aspect,the cells to be analyzed are cultured with an accelerated growthkinetics by at least 100%, 200% or 300% or more as compared to the plainsurface substrate of the same composition but without the said nanotubestructure. In one aspect, the diagnosis and detection device is anoptical detection device, a chemical or biological detection device, ora magnetic sensor device.

The invention provides analytical cell diagnostic biochip systems wherethe cells to be detected and counted are diseased cells or cells exposedto toxins, poisons, biological agents, e.g., biological warfare agents,e.g., bacteria or viruses, or are forensic test-related cells. In oneaspect, the growth and proliferation of cells to be detected and countedare further accelerated by controlled or externally initiated release ofbiological or functional agents stored in the reservoir of nanotubes. Inone aspect, the biochips are arranged in an X-Y matrix configurationcomprising a subdivided array of TiO.sub.2 nanotube regions forsimultaneous fast diagnosis and detection of diseased cells or cellsexposed to toxins, poisons, biological agents, e.g., biological warfareagents, e.g., bacteria or viruses, or, are forensic test-related cells.

The invention provides methods of fabricating in vitro or in vivo cellgrowth devices (e.g., accelerated cell growth devices) and implants,orthopaedic or dental implant structures, and disease or toxin celldetection devices, forensic cell detection, drug toxicity testingdevices, artificial liver devices, artificial kidney devices, all havingvertically aligned surface nanotube structures, e.g., by usingelectrochemical anodization of metallic base implant material, e.g., Ti,Zr, Hf, Nb, Ta, Mo, W and/or alloys of these metals, or alloys withother metals having the alloy contents of less than 10%, 20%, 30%, 40%50% or more weight % total.

The invention provides methods of fabricating in vitro or in vivo cellgrowth devices (e.g., accelerated cell growth devices) and implants,orthopaedic or dental implant structures, and disease cell detectiondevices, forensic cell detection, drug toxicity testing devices,artificial liver devices, artificial kidney devices, all havingvertically aligned surface nanotube structure by (a) preparing alaterally separated nanotube array structure by lithographic, chemical,electrochemical or reactive ion etch process on ceramics, Si, Si oxide,carbon, diamond, Au, Pt and their alloys, polymer or plastic materials,ceramic or composite metals. (b) adding a metallic or oxide coating bythin film sputter deposition, evaporation, or CVD deposition of Ti, Zr,Hf. Nb, Ta, Mo, W, or alloys of these metals on the surface of thefabricated nanotube structure, and in some aspects (c) with a coatingthickness of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nm andthe coating coverage of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%or 90% of the total surfaces, and in some aspects (d) the depositedmetal is converted to oxide by exposure to oxidizing atmosphere atambient or heated temperature, by chemical process or by electrochemicaloxidation process.

The invention provides methods of fabricating in vitro or in vivo cellgrowth devices (e.g., accelerated cell growth devices) and orthopaedicor dental implant structures of the invention further comprisingmulti-functional biological agents incorporated into the nanotubereservoirs, e.g., by ultrasonication. In one aspect, themulti-functional biological agents are incorporated into the nanotubereservoirs by heating the nanotube structure in boiling water to removethe trapped air bubble in the nanotubes.

The invention provides devices. e.g., bio-assemblies, comprisingthree-dimensionally and cultured (e.g., rapidly cultured) animal orhuman cells, tissues, organs and bones attached and grown onthree-dimensionally configured and closely spaced biocompatible metallicribbons and wires, wherein (a) the surface of the metallic elements havevertically and parallel-aligned laterally-separated nanotube or nanoporearray structure, and in one aspect (b) the rapidly cultured cells,tissues and bones have an accelerated growth kinetics by at least 100%,200%, 300% or more as compared to the plain metal wire or ribbon surfacewithout the said nanotube or nanopore structure, and in one aspectcomprising (c) metallic wires and ribbons made of a material comprisingTi, Zr, Hf. Nb, Ta, Mo, W and/or their alloys with each other orcontaining other alloying elements of less than 10, 20, 30, 40 or 50 ormore weight percent total, and in one aspect (d) the nanotube array orthe surface of the nanopore array material comprises an oxides materialcomprising Ti oxide, Zr oxide, Hf oxide, Nb oxide, Ta oxide, Mo oxide, Woxide, and/or mixed oxide alloyed with each other or containing otheralloying element oxides of less than 10, 20, 30, 40 or 50 or more weightpercent total. In one aspect, the metallic ribbons and wires areparallel and linearly arranged for easy pull out from the assembly. Inone aspect, the metallic ribbons and wires are curved, bent or tangledfor mechanical lock-in, reinforcement and support of grown cells.

In one aspect, the rapidly cultured cells, tissues and bones compriseliver cells, kidney cells, nerve cells, myocytes, stem cells, supportivesoft tissues such as muscles, tendons, fibrous tissues, skin cells,periodontal tissues, fat, blood vessels, and hard tissues such as boneand teeth, either as a single type cell culture or as a co-culture aco-culture of at least two types of cells together.

In one aspect, the metallic ribbons and wires are subdivided and have apreferred dimension of (a) a thickness of in the range of between about25 to 2500 micrometers, or 50 to 500 micrometers. In one aspect, spacingbetween the parallel neighboring branches of metallic wires and ribbonsin the cell cultured assembly is at most 5, 10, 15, 20 or more times thethickness of an average monolayer cell thickness, or between at most 5to 10 times the thickness of an average monolayer cell.

In one aspect, the surface of the ribbons and wires contains an alignednanopore or nanotube array with the preferred diameter being in therange of between about 10-1000 nm, or 50-500) nm, and in one aspect, thedesired height being in the range of between about 40-2000 nm, or100-400 nm, and in one aspect the desired angle is vertical with anallowance of 10, 20 or 30 degree variation off the perpendicular axis,and in one aspect, the desired volume fraction of the metal for thegiven targeted cell volume at the end of the planned cell culture periodis at least 10%, 20%, 300/%, 40% or 50% or more.

In alternative aspects, the bio-assembly comprises an orthopedic, dentalor periodontal implant, or an artificial organ, or comprises liver cellsand the assembly is a liver or kidney implants, or comprises liver cellsand the assembly is an artificial liver device, or comprises liver cellsand the assembly is a drug toxicity testing device, or is a celldetection device for rapidly identifying disease, forensic or biologicalwarfare agent-exposed cells, or is a device for rapid cell culture,detachment and supply.

In alternative aspects, biocompatible vertically aligned nanotube arraystructures are strongly adhered on a metallic wire or ribbon substrate,and can have an outer diameter of nanotubes is in the range of betweenabout 10-1000 nm, or an alignment orientation in the range of betweenabout 0-30 degrees off the vertical direction from the wire or ribbonsurface, or the desired lateral spacing between adjacent nanotubes canbe in the range of between about 2-100 nm.

In one aspect, nanotubes on the surface of metallic wires and ribbonsare made by electrochemical anodization process. Nanotubes on thesurface of metallic wires and ribbons also can be made by artificialpattern design and lithography.

The invention provides three-dimensionally and rapidly cultured cells,tissues, organs and bones wherein (a) the surface of the said metallicelements have vertically and parallel-aligned laterally-separatednanotube or nanopore array structure, (b) the cells are attached andgrown on three-dimensionally configured and closely spaced biocompatiblemetallic ribbons and wires, (c) the metallic ribbons and wires areparallel and linearly arranged for rapid cell growth, and then pulledout from the cultured three-dimensional cell assembly so that the cellassembly has no metallic or ceramic substrate left, and in one aspect(d) the substrate-free cell assembly is allowed to heal itself to fillup the space left by the retracted metallic wires and ribbons, and inone aspect (e) the rapidly cultured cells, tissues and bones have anaccelerated growth kinetics by at least 100%6, 200%, 300% or more ascompared to the plain metal wire or ribbon surface without the saidnanotube or nanopore structure, and in one aspect (f) the metallic wiresand ribbons are made of a material comprising Ti, Zr, Hf, Nb, Ta. Mo. W,and/or their alloy with each other and/or comprising other alloyingelements of less than 10, 20, 30, 40, 50 or more weight percent total,and in one aspect (g) the nanotube array or the surface of the nanoporearray material comprising an oxides material comprising Ti oxide, Zroxide, Hf oxide, Nb oxide. Ta oxide, Mo oxide, W oxide, and mixed oxidealloyed with each other or comprising other alloying element oxides ofless than 10, 20, 30, 40, 50 or more weight percent total.

In one aspect, the in vivo or in vitro nutrient fluid is supplied underthe growing cells through the gap spacing between the nanotubes inaddition to the overall space including the top of the cells. In oneaspect, the vertical pores of the nanotubes or nanotubes serve as areservoir and contain various biologically active agents such astherapeutic drugs, growth factors, proteins, collagens, stem cells,enzymes, hormones, nucleic acids (e.g., RNA, DNA, genes, vectors),antibiotics, antibodies, magnetic nanoparticles, radioisotopes and othermaterials for slow or externally actuated release.

The invention provides controlled-release drug and functional agentdelivery devices comprising the structure of three-dimensionallyconfigured and closely spaced biocompatible metallic ribbons and wireswherein (a) the surface of the said metallic elements have verticallyand parallel-aligned laterally-separated nanotube or nanopore arraystructure, (b) the said metallic wires and ribbons are made of amaterial selected from a list of Ti, Zr, Hf, Nb, Ta, Mo. W, and theiralloy with each other or containing other alloying elements of less than10, 20, 30, 40, 50 or more weight percent total, (c) the nanotube arrayor the surface or the nanopore array material is an oxides materialselected from a list of Ti oxide, Zr oxide, Hf oxide, Nb oxide, Taoxide, Mo oxide, W oxide, and mixed oxide alloyed with each other orcontaining other alloying element oxides of less than 10, 20, 30, 40, 50or more weight percent total, and optionally coated with Ti or Ti oxidefilm of at least 5 nm thickness and the coating coverage of at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the total surfaces; andin one aspect (d) the biocompatible aligned nanotube or nanotube arraystructure is strongly adhered on a metallic substrate, and has thedesired outer diameter of nanotubes is in the range of 10-1000 nm, thedesired range of variation in the vertical alignment angle within 0-30degrees off the vertical direction, and the desired lateral spacingbetween adjacent nanotubes in the range of 2-100 nm; and in one aspect(e) wherein the inside pores of the said vertically aligned nanotubesare reservoirs containing biologically active agents including at leastone of the therapeutic drugs, growth factors, proteins, enzymes,hormones, nucleic acids (e.g., RNA, DNA, genes, vectors), antibiotics,antibodies, magnetic nanoparticles, radioisotopes and other materials,or functionally active particles such as magnetic nanoparticles, novelmetal nanoparticles or ceramic nanoparticles; and in one aspect (f)wherein the drug release device is implanted in a living body andtherapeutic drugs are slowly released from the nanotubes at a ratedetermined by the nanotube diameter and aspect ratio, and the viscosityand wetting characteristics of the drug-containing solution storedwithin the nanotubes.

The invention provides methods of fabricating in vitro or in vivo cellgrowth devices and implants, orthopedic or dental implant structures,and disease cell detection devices, forensic cell detection, drugtoxicity testing devices, artificial liver devices, artificial kidneydevices with a structure of the invention by (a) first arranging metalwires or ribbons into a three-dimensional configuration, (b) thenconverting their surfaces into a vertically aligned nanotube or nanoporestructure by using electrochemical anodization of metallic base implantmaterial comprising Ti, Zr, Hf, Nb, Ta, Mo. W, or alloys of these metalsamong them or alloys with other metals having the alloy contents of lessthan 50% weight % total.

The invention provides methods of fabricating in vitro or in vivo cellgrowth devices and implants, orthopedic or dental implant structures,and disease cell detection devices, forensic cell detection, drugtoxicity testing devices, artificial liver devices, artificial kidneydevices with a structure of the invention, having vertically alignedsurface nanotube structure by (a) preparing a laterally separatednanotube array structure by lithographic, chemical, electrochemical orreactive ion etch process on ceramics. Si. Si oxide, carbon, diamond.Au, Pt and their alloys, polymer or plastic materials, ceramic orcomposite metals, (b) adding a metallic or oxide coating by thin filmsputter deposition, evaporation, or CVD deposition of Ti, Zr, Hf, Nb,Ta. Mo. W, or alloys of these metals on the surface of the fabricatednanotube structure, and in one aspect (c) with a coating thickness of atleast 1, 2, 3, 4 or 5 nm and the coating coverage of at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 8(0% or 90% of the total surfaces, and in oneaspect, (d) the deposited metal optionally converted to oxide byexposure to oxidizing atmosphere at ambient or heated temperature, bychemical process or by electrochemical oxidation process.

The invention provides methods of fabricating in vitro or in vivo cellgrowth devices and implants, and orthopedic or dental implantstructures, wherein multi-functional biological agents of the inventionare incorporated into the nanotube reservoirs by ultrasonication.

The invention provides methods of fabricating in vitro or in vivo cellgrowth devices and implants, and orthopedic or dental implant structuresof the invention, wherein multi-functional biological agents of theinvention are incorporated into the nanotube reservoirs by heating thenanotube structure in boiling water to remove the trapped air bubble inthe nanotubes.

The invention provides bio-assemblies comprising rapidly cultured animalor human cells, tissues, organs and bones attached and grown ondual-structured, biocompatible substrates with both micro and nanotopological configurations, wherein (a) the micro configurations have anaverage diameter (or equivalent diameter if the pores are not circular)in the range of between about 0.5-1,000.mu.m, or 1-100.mu.m. (b) thesurface of the said substrates have nano configurations of verticallyand parallel-aligned laterally-separated nanotube or nanopore arraystructure with the average nanopore or nanotube diameter in thepreferred range of e.g., 30-600 nm, (c) the relative ratio of the micropores vs nano pores in the dual structure is such that the micro/macropores desirably occupy at least 10/o, 20%, 30%, 40%, 50%, 60% o, 70%,80% or 90% area fraction of the implant or bio-substrate surface, or atleast 20% but less than 50%, to maximize the accelerated cell culture orbone growth via the nanopore portion of the surface; and in one aspect(d) the rapidly cultured cells, tissues and bones are selected from alist of functional cells such as liver cells, kidney cells, nerve cells,myocytes, stem cells, supportive soft tissues such as muscles, skincells, tendons, fibrous tissues, periodontal tissues, fat, bloodvessels, and hard tissues such as bone and teeth, either as a singlecell type culture or as a co-culture of at least two types of cellstogether; and in one aspect (e) the rapidly cultured cells, tissues andbones have an accelerated growth kinetics by at least 100%, 200%, 300%or more as compared to the same substrate without the said nanotube ornanopore structure; and in one aspect (f) the said substrate is made ofa material selected from a list of Ti, Zr, Hf. Nb, Ta, Mo, W, and theiralloy with each other or containing other alloying elements of less than10, 20, 30, 40 or 50 weight percent total; and in one aspect (g) thenanotube array or the surface of the nanopore array material comprisesan oxides material comprising Ti oxide, Zr oxide, Hf oxide, Nb oxide, Taoxide, Mo oxide, W oxide, and/or mixed oxide alloyed with each other orcontaining other alloying element oxides of less than 50 weight percenttotal.

In one aspect, the micro- or nano-features have a lock-in structure witha re-entrant configuration with the pore or nanopore entrance having asmaller diameter than the rest of the pore or nanopore dimension so thatthe cells or bones grown are mechanically more firmly attached, wherein;the re-entrant feature can be either circular, oval, tapered wall, orcorrugated shape, the degree of the maximum re-entrance is at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the largest diameter of thepore or nanopore.

The invention provides methods of fabricating a lock-in structure with are-entrant pore or nanopore configuration in a bio-assembly of theinvention; and in one aspect the re-entrant configuration is obtained byoblique incident physical vapor deposition; and in one aspect, there-entrant configuration is obtained by gradual increase ofelectrochemical anodization voltage; and in one aspect, the re-entrantconfiguration is obtained by a use of isotropic etching on a patternmasked substrate.

In one aspect, the nanopores in the bio assembly serve as a reservoirand contain at list one of various chemicals, substances and/orbiologically active agents, e.g., therapeutic drugs, growth factors,proteins, collagens, stem cells, enzymes, hormones, nucleic acids,antibiotics, antibodies, magnetic, metallic, ceramic, polymer, or bioimaging nanoparticles.

In alternative aspects, the dual-structured bio-assemblies areorthopedic or dental implants, artificial organs, or comprise livercells and the assembly is a liver implants, or comprise liver cells andthe assembly is an artificial liver device, or comprises liver cells andthe assembly is a drug toxicity testing device, or is a cell detectiondevice for rapidly identifying a disease or conditions, e.g., a diseasedcell, or a cell exposed to a biological or chemical warfare agent, or acell useful in forensic purposes, or is a device for rapid cell culture,detachment and supply.

In alternative aspects, the dual-structured bio-assemblies comprisecells and/or bones cultured in a two- and/or three-dimensionalconfiguration.

The dual-structured bio-assemblies can comprise coated Si, Si oxide,carbon, diamond, noble metals (such as Au, Ag, Pt and their alloys),polymer or plastic materials, or composite metals, ceramics or polymershaving a laterally separated nanotube configuration and having apreferential surface coating with Ti oxide, Ti alloy oxide, or an oxideof Zr, Hf, Nb, Ta, Mo, W and their alloys, and in one aspect (c) with acoating thickness of at least 1, 2, 3, 4 or 5 nm and the coatingcoverage of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% ofthe total surfaces, and in one aspect, (d) the deposited metaloptionally converted to oxide by exposure to oxidizing atmosphere atambient or heated temperature, by chemical process or by electrochemicaloxidation process.

In one aspect, the invention provides methods for fabricating thedual-structured bio-assemblies by using a combination of either photolithography and/or electrochemical anodization, or, by using acombination of nano imprint lithography and electrochemical anodization.In one aspect, the nano-imprint lithography utilizes a compliantelastomeric mask sheet or an elastomeric stamp. In one aspect, thedual-structured bioassembly is fabricated using a combination of guidedetching using a vertically two-phase decomposed coating andelectrochemical anodization. In one aspect, the dual-structuredbioassembly is fabricated using a combination of guided etching using avertically two-phase decomposed coating of periodically or spinodallydecomposing alloy and electrochemical anodization.

In one aspect, the bio-assembly comprises rapidly cultured animal orhuman cells, tissues, organs and bones attached and grown onenlarged-diameter, surface nanotube array on biocompatible substrateswherein (a) the diameter-enlarged surface nanotube array has avertically and parallel-aligned and laterally-separated nanotubeconfiguration, and has an average nanotube diameter in the preferredrange of at least 50, 100, 150, 200, 250, 300 or more nm diameter. Inone aspect, the cultured (e.g., rapidly cultured) cells, tissues andbones are selected from a list of functional cells such as liver cells,kidney cells, nerve cells, myocytes, stem cells, supportive soft tissuessuch as muscles, skin cells, tendons, fibrous tissues, periodontaltissues, fat, blood vessels, and hard tissues such as bone and teeth,either as a single cell type culture or as a co-culture of at least twotypes of cells together. In one aspect, the cultured (e.g., rapidlycultured) cells, tissues and bones have an accelerated growth kineticsby at least 100%, 200%, 300% or more as compared to the same substratewithout the said nanotube or nanopore structure. In one aspect, thesubstrate is made of a material comprising Ti, Zr, Hf; Nb, Ta. Mo, W,and/or their alloy with each other or containing other alloying elementsof less than 10, 20, 30, 40 or 50 weight percent total. In one aspect,the nanotube array or the surface of the nanopore array materialcomprises an oxides material selected from a list of Ti oxide, Zr oxide,Hf oxide, Nb oxide. Ta oxide, Mo oxide, W oxide, and mixed oxide alloyedwith each other or containing other alloying element oxides of less than10, 20, 30, 40 or 50 weight percent total.

The invention provides bio-assemblies comprising cultured (e.g., rapidlycultured) cells, e.g., cultured animal or human cells, tissues, organsand/or bones, attached and grown on randomized-diameter, surfacenanotube array on biocompatible substrates wherein; (a) the saidrandomized-diameter, surface nanotube array has a vertically andparallel-aligned and laterally-separated nanotube configuration, and hasa desired distribution of the nanopore sizes so that at least one thirdof the pores have their average diameter which is equal to or less than70%, 60% 50%, or 40%, or equal to or less than about 50% of the overallaverage pore diameter, while another one third of the pores have theiraverage diameter which is at least 125%, 150% or 200% or more or theoverall average pore diameter and optionally the distribution of thenanopore or nanotube sizes is such that at least one quarter to one halfof the pores or tubes have their average diameter equal to or less than20% to 80% of the overall average pore diameter, while another onequarter to one half of the pores have their average diameter at least100% to 150%, or 100% to 200%, of the overall average pore diameter.

In one aspect of the diameter-enlarged bio assembly of the invention orthe diameter-randomized bio assembly of the invention, the nanopores inthe biocompatible substrate have a lock-in structure with a re-entrantconfiguration with the pore or nanopore entrance having a smallerdiameter than the rest of the pore or nanopore dimension so that thecells or bones grown are mechanically more firmly attached, wherein (a)the re-entrant feature can be either circular, oval, tapered wall, orcorrugated shape, and/or (b) the degree of the maximum re-entrance is atleast 5% or 10% or more of the largest diameter of the pore or nanopore.

In one aspect, the invention provides methods for fabricating adiameter-randomized nanopore- or nanotube-comprising device, e.g., abio-assembly device of the invention, by guided patterning of substratesurface using a roll stamping of elastomeric nano implant stamp tocreate an etch mask of diameter-randomized pattern, or, by guidedpatterning of substrate surface using a large-area elastomeric nanostamping in combination with reactive ion etch or chemical etch.

In one aspect, the invention provides methods for fabricating thecontrolled-release drug and/or functional agent delivery devices of theinvention having a diameter-enlarged and/or a diameter-randomizedsubstrate surface, comprising nanostructures wherein (a) the surface ofthe substrate has vertically and/or parallel-aligned laterally-separatednanotube or nanopore array structure; and in alternative aspects (b) thesubstrate is made of a material comprising Ti, Zr, Hf, Nb, Ta, Mo, W,and their alloy with each other or containing other alloying elements ofless than 10, 20, 30, 40 or 50 weight percent total; and in alternativeaspects (c) the nanotube array or the surface of the nanopore arraymaterial comprises an oxides material comprising Ti oxide, Zr oxide, Hfoxide, Nb oxide, Ta oxide, Mo oxide, W oxide, and/or mixed oxide alloyedwith each other and/or containing other alloying element oxides of lessthan 10, 20, 30, 40 or 50 weight percent total, and optionally coatedwith Ti or Ti oxide film of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ormore nm thickness: and in alternative aspects a coating coverage of atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the totalsurfaces; and in alternative aspects (d) the biocompatible alignednanotubes or nanopores array structure is strongly adhered on a metallicsubstrate; and in alternative aspects has the desired outer diameter ofnanotubes is in the range of between about 10-1000 nm, the desired rangeof variation in the vertical alignment angle within between about 0-30degrees off the vertical direction, and the desired lateral spacingbetween adjacent nanotubes in the range of between about 2-100 nm.

In one aspect, the inside pores of the said vertically aligned nanotubesor nanopores are reservoirs comprising any desired substance, e.g., achemical, an agent (e.g., a radioisotope, dye), a biologically activeagent, e.g., therapeutic drugs, growth factors, proteins, enzymes,hormones, nucleic acids, antibiotics, or antibodies, or functionallyactive particles such as magnetic nanoparticles, novel metalnanoparticles or ceramic nanoparticles. In one aspect, the drug releasedevice is implanted in a living body and therapeutic drugs are slowlyreleased from the nanotubes or nanopores at a rate determined by thenanotube diameter and aspect ratio, and the viscosity and wettingcharacteristics of the drug-containing solution stored within thenanotubes. In one aspect, the drugs stored in the nanotubes or nanoporescomprise cancer treatment drugs, diabetes drugs, bone-growthaccelerating drugs, tissue-formation-preventing drugs or anti-stenosisdrugs.

In one aspect, for the controlled-release drug and/or functional agentdelivery devices of the invention having a diameter-enlarged and/or adiameter-randomized nanotube- or nanopore-comprising substrate surface,the structure of the nanotube or nanopore ends are of partially cappedconfiguration to enhance retaining the desired composition, e.g., smallmolecule, biological or other functional agents within the nanotube ornanopore. In one aspect, the drug release is externally controllable sothat the timing as well as the quantity of the release of drugs orbiological agents is initiated and stopped at will by ultrasonic ormagnetic agitation of the colloidal liquid containing the mixture of thedrug solution and the nanoparticles, as discussed above.

The invention provides analytical cell diagnostic biochip systemscomprising a cell culture medium, the intended cells to be analyzed, acell growth substrate comprising a diameter-enlarged bioassembly of theinvention or the diameter-randomized bioassembly of the invention, andcell growth monitoring detection device wherein the diagnosis anddetection device, e.g., an optical detection device, a chemical orbiological detection device and/or magnetic sensor device. In oneaspect, the cells to be detected and counted are diseased cells, cellsexposed to biological or chemical warfare agents, toxins, poisons, orforensic-test-related cells. In one aspect, the growth and proliferationof cells to be detected and counted are further accelerated bycontrolled or externally initiated release of biological or functionalagents stored in the reservoir of nanotubes.

It should be understood that the invention can be practiced withmodification and alteration within the spirit and scope of the appendedclaims. The description is not intended to be exhaustive or to limit theinvention to the precise form disclosed. It should be understood thatthe invention can be practiced with modification and alteration and thatthe invention be limited only by the claims and the equivalents thereof.

1: A biocompatible nanotube array structure comprising a plurality ofnanotubes, wherein: an outer diameter of each nanotube is between about10-1000 nanometers (nm); an inside diameter of each nanotube is about10% to 50% of the outer diameter; a height of each nanotube is betweenabout 40-800 nm; an aspect ratio is less than about 10; a verticalalignment angle is within about 0-45 degrees off the vertical direction;and lateral spacing between adjacent nanotubes is about between 2-100nm. 2: The biocompatible nanotube array structure of claim 1, whereinthe biocompatible nanotube array structure comprises a verticallyaligned titanium oxide nanotube array structure on a titanium ortitanium oxide substrate. 3: The biocompatible nanotube array structureof claim 2, wherein sodium titanate nanostructures are superimposed ontothe vertically aligned titanium oxide nanotube array structure. 4: Thebiocompatible nanotube array structure of claim 1, wherein an insidepore of each nanotube comprises at least one biologically active agentselected from the group consisting of pharmaceutical compositions,therapeutic drugs, growth factors, proteins, enzymes, hormones, nucleicacids, antibiotics and antibodies. 5: The biocompatible nanotube arraystructure of claim 4, wherein the inside pore comprises magneticnanoparticles. 6-7. (canceled) 8: An orthopedic implant comprising thebiocompatible nanotube array structure of claim 1, wherein a surface ofthe orthopedic implant comprises an adherent titanium oxide nanotubearray. 9: A dental implant comprising the biocompatible nanotube arraystructure of claim 1, wherein a surface of the dental implant comprisesan adherent titanium oxide nanotube array. 10: A multi-functionalimplant device comprising the biocompatible nanotube array structure ofclaim 1, wherein a vertical pore of each nanotube contains a reservoirof biologically active agents selected from the group consisting ofpharmaceutical compositions, therapeutic drugs, cancer drugs, growthfactors, proteins, enzymes, hormones, nucleic acids, antibiotics,antibodies, nanoparticles, and a biologically active material. 11: Themulti-functional implant device of claim 10, wherein themulti-functional implant device is designed for externally controlledrelease of a colloidal liquid upon application of ultrasonic or magneticstimulation. 12: The multi-functional implant device of claim 10,wherein a cap is deposited at an upper end of each nanotube by anoblique incident sputter deposition on a stationary or a rotatingsubstrate. 13-14. (canceled) 15: A method of cell proliferationcomprising the biocompatible nanotube array structure of claim 1, andadherent cells, wherein upon adhesion the adherent cells are induced toproliferate. 16-17. (canceled) 18: A method for producing abiocompatible nanotube array structure, the method comprising: providinga structure comprising vertically aligned, biocompatible titanium oxidenanotubes having dimensions of between 10 to 100 nm outer diameter, andbetween 10 to 90 nm inner diameter, and between 10 to 100 nm wallthickness, and between 20 to 300 nm in height; fabricating a titaniumoxide nanotube array structure by anodization technique using a titaniumsheet, the anodization technique including electrochemically processingthe titanium sheet in a 0.5% HF solution at an applied voltage ofbetween 10-30 V for between 5-200 min; and crystallizing depositedamorphous-structure titanium nanotubes into an anatase phase byheat-treating the titanium oxide nanotube array structure at between4500 C to 5500 C for between about 0.1-24 hrs. 19: A lock-innanostructure comprising a plurality of nanotubes, wherein each nanotubeentrance of the plurality of nanotubes has a smaller diameter than aninterior of the nanotube to exhibit a re-entrant configuration. 20: Thelock-in nanostructure of claim 19, wherein each nanotube entrance has anaverage entrance diameter at least 10% to 50% smaller than the interiorof the nanotube. 21: The lock-in nanostructure of claim 19, wherein thenanostructure comprises Ti or TiO2 in a nanotube configuration. 22: Adual structured biomaterial comprising: micropores, wherein themicropores have an average diameter, or equivalent diameter if themicropores are not circular, between 0.5-1,000 μm; and a surface areacovered with nanotube each having an average pore diameter between30-600 nm. 23: The dual structured biomaterial of claim 22, wherein thedual structured biomaterial comprises any of an implant, an artificialorgan, and an artificial joint. 24: A biomaterial, comprising: aplurality of enlarged diameter nanotubes, wherein each of the pluralityof enlarged diameter nanotubes has a diameter of at least about 150 nm;and any of a biological agent, a small molecule, a growth factor, acollagen, a nucleic acid, an antibiotic, a hormone, a drug, a magneticparticle, a metallic particle, a radioisotope, a ceramic particle, apolymer particle, a drug delivery particle, a lipid, a liposome, acarbohydrate, a nucleic acid or a combination thereof.
 25. (canceled)26: The biomaterial of claim 24, wherein the biomaterial furthercomprises any of an implant, an artificial organ, and an artificialjoint. 27: The biomaterial of claim 24, wherein the biomaterial furthercomprises a plurality of cells. 28-119. (canceled)